Tailored oils produced from recombinant heterotrophic microorganisms

ABSTRACT

Methods and compositions for the production of oil, fuels, oleochemicals, and other compounds in recombinant microorganisms are provided, including oil-bearing microorganisms and methods of low cost cultivation of such microorganisms. Microalgal cells containing exogenous genes encoding, for example, a lipase, a sucrose transporter, a sucrose invertase, a fructokinase, a polysaccharide-degrading enzyme, a keto acyl-ACP synthase enzyme, a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, and/or an acyl carrier protein are useful in manufacturing transportation fuels such as renewable diesel, biodiesel, and renewable jet fuel, as well as oleochemicals such as functional fluids, surfactants, soaps and lubricants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/118,365, filed May 27, 2011, which claims the benefit under 35 U.S.C.119(e) of U.S. Provisional Patent Application No. 61/349,774, filed May28, 2010, U.S. Provisional Patent Application No. 61/374,992, filed Aug.18, 2010, U.S. Provisional Patent Application No. 61/414,393, filed Nov.16, 2010, and U.S. Provisional Patent Application No. 61/428,192, filedDec. 29, 2010. Each of these applications is incorporated herein byreference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“422205-Sequence.txt”, created on Jul. 6, 2012 and containing 1,006,168bytes, which is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to the production of oils, fuels, andoleochemicals made from microorganisms. In particular, the disclosurerelates to oil-bearing microalgae, methods of cultivating them for theproduction of useful compounds, including lipids, fatty acid esters,fatty acids, aldehydes, alcohols, and alkanes, and methods and reagentsfor genetically altering them to improve production efficiency and alterthe type and composition of the oils produced by them.

BACKGROUND OF THE INVENTION

Fossil fuel is a general term for buried combustible geologic depositsof organic materials, formed from decayed plants and animals that havebeen converted to crude oil, coal, natural gas, or heavy oils byexposure to heat and pressure in the earth's crust over hundreds ofmillions of years. Fossil fuels are a finite, non-renewable resource.Increased demand for energy by the global economy has also placedincreasing pressure on the cost of hydrocarbons. Aside from energy, manyindustries, including plastics and chemical manufacturers, rely heavilyon the availability of hydrocarbons as a feedstock for theirmanufacturing processes. Cost-effective alternatives to current sourcesof supply could help mitigate the upward pressure on energy and theseraw material costs.

PCT Pub. No. 2008/151149 describes methods and materials for cultivatingmicroalgae for the production of oil and particularly exemplifies theproduction of diesel fuel from oil produced by the microalgae Chlorellaprotothecoides. There remains a need for improved methods for producingoil in microalgae, particularly for methods that produce oils withshorter chain length and a higher degree of saturation and withoutpigments, with greater yield and efficiency. The present invention meetsthis need.

SUMMARY OF THE INVENTION

The present invention provides oleaginous microbial cells, preferablymicroalgal cells, having distinct lipid profiles, and includesrecombinant cells expressing exogenous genes encoding proteins such asfatty acyl-ACP thioesterases. The present invention also providesmethods of making lipids and oil-based products, including fuels such asbiodiesel, renewable diesel and jet fuel, from such cells.

In a first aspect, the present invention provides oleaginous microbialcells, preferably microalgal cells, having a lipid profile that is atleast 1% or at least 5%, preferably at least 3%, C8:0. In some cases,the lipid profile is at least 10% or at least 15%, preferably at least12%, C8:0. In some embodiments, the cell is a recombinant cell. In somecases, the recombinant cell comprises an exogenous gene encoding anacyl-ACP thioesterase protein that has hydrolysis activity towards fattyacyl-ACP substrates of chain length C8. In some embodiments, theexogenous gene encodes a Cuphea palustris acyl-ACP thioesterase. In somecases, the cell is a Prototheca cell. In some cases, the cell is of amicroalgal genus or species selected from microalgae identified in Table1.

In a second aspect, the present invention provides oleaginous microbialcells, preferably microalgal cells, having a lipid profile that is atleast 4% C10:0. In some cases, the lipid profile is at least 20%, atleast 25% or at least 30%, preferably at least 24%, C10:0. In somecases, the ratio of C10:0 to C12:0 is at least 6:1. In some embodiments,the cell is a recombinant cell. In some cases, the recombinant cellcomprises an exogenous gene encoding an acyl-ACP thioesterase proteinthat has hydrolysis activity towards fatty acyl-ACP substrates of chainlength C10. In some embodiments, the exogenous gene encodes an acyl-ACPthioesterase protein from a species selected from the group consistingof Cuphea hookeriana and Ulmus americana. In some cases, the cell is aPrototheca cell. In some embodiments, the cell is of a microalgal genusor species selected from microalgae identified in Table 1.

In a third aspect, the present invention provides oleaginous microbialcells, preferably microalgal cells, having a lipid profile that is atleast 10% or at least 15%, preferably at least 13%, C12:0. In somecases, the lipid profile is at least 30%, at least 35% or at least 40%,preferably at least 34%, C12:0. In some cases, the ratio of C12 to C14is at least 5:1. In some cases, the cell is a recombinant cell. In someembodiments, the recombinant cell comprises an exogenous gene encodingan acyl-ACP thioesterase protein that has hydrolysis activity towardsfatty acyl-ACP substrates of chain length C12. In some cases, therecombinant cell comprises at least two exogenous genes encodingacyl-ACP thioesterase proteins from Umbellularia californica andCinnamomum camphora that have hydrolysis activity towards fatty acyl-ACPsubstrates of chain length C12. In some embodiments, the cell is aPrototheca cell.

In a fourth aspect, the present invention provides oleaginous microbialcells, preferably microalgal cells, having a lipid profile that is atleast 5% or at least 15%, preferably at least 10%, C14:0. In some cases,the lipid profile is at least 40%, at least 45%, or at least 50%,preferably at least 43%, C14:0. In some cases, the ratio of C14:0 toC12:0 is at least 7:1. In some cases, the cell is a recombinant cell. Insome embodiments, the recombinant cell comprises an exogenous geneencoding an acyl-ACP thioesterase protein that has hydrolysis activitytowards fatty acyl-ACP substrates of chain length C14. In someembodiments, the acyl-ACP thioesterase protein is from a speciesselected from the group consisting of Cinnamomum camphora and Ulmusamericana. In some cases, the cell is a Prototheca cell. In someembodiments, the cell is of a microalgal genus or species selected frommicroalgae identified in Table 1.

In a fifth aspect, the present invention provides oleaginous microbialcells, preferably microalgal cells, having a lipid profile that is atleast 10% or at least 20%, preferably at least 15%, C16:0. In somecases, the lipid profile is at least 30%, at least 35% or at least 40%,preferably at least 37%, C16:0. In some cases, the cell is a recombinantcell. In some embodiments, the recombinant cell comprises an exogenousgene encoding an acyl-ACP thioesterase protein that has hydrolysisactivity towards fatty acyl-ACP substrates of chain length C16. In someembodiments, the recombinant cell comprises at least two exogenous genesencoding acyl-ACP thioesterase proteins from Umbellularia californicaand Cinnamomum camphora that have hydrolysis activity towards fattyacyl-ACP substrates of chain length C16. In some cases, the cell is aPrototheca cell.

In a sixth aspect, the present invention provides oleaginous microbialcells, preferably microalgal cells, having a lipid profile that is atleast 55% or at least 65%, preferably at least 60%, saturated fattyacids. In some cases the cells, have a lipid profile that is at least80%, at least 85%, or at least 90%, preferably at least 86%, saturatedfatty acids. In some cases, the cell is a recombinant cell. In someembodiments, the recombinant cell comprises an exogenous gene encodingan acyl-ACP thioesterase protein that has hydrolysis activity towardsfatty acyl-ACP substrates of chain lengths C10-C16. In some embodiments,the cell comprises an exogenous gene encoding a ketoacyl synthaseprotein. In some cases, the cell is a Prototheca cell.

In a seventh aspect, the present invention provides oleaginous microbialcells, preferably microalgal cells, comprising a mutated endogenousdesaturase gene, wherein the mutation renders the gene or desaturaseinactive. In some cases, the cell has a lipid profile that is at least40% or at least 50%, preferably at least 45%, saturated fatty acids. Insome cases, the cell has a lipid profile that is at least 15%, at least20% or at least 25%, preferably at least 19%, C18:0. In someembodiments, the cell comprises a mutated endogenous desaturase genethat results in at least a 2-fold increase in C18:0 fatty acid, ascompared to a wild-type cell. In some cases, the microalgal cell has alipid profile that is no more than 1% or no more than 5%, preferably nomore than 2%, C18:2. In some embodiments, the microalgal cell has alipid profile that is no more than 5% or no more than 10%, preferably nomore than 7%, 18:1.

In some embodiments of the recombinant cells discussed herein, the cellcomprises a mutated endogenous desaturase gene, wherein the mutationrenders the gene or desaturase inactive.

In a eighth aspect, the present invention provides a method of makinglipid. In one embodiment, the method comprises (a) cultivating a cell asdiscussed above until the cell is at least 15% or at least 25%,preferably at least 20%, lipid by dry weight, and (b) separating thelipid from water-soluble biomass components.

In a ninth aspect, the present invention provides another method ofmaking lipid. In one embodiment, the method comprises (a) cultivating anoleaginous microbial, preferably a microalgae cell, containing exogenousgenes encoding two distinct acyl-ACP thioesterases, wherein the lipidprofile of the cell is distinct from (i) the profile of the cell withoutthe exogenous genes and (ii) the profile of the cell with only one ofthe exogenous genes, and (b) separating the lipid from water-solublebiomass components. In some cases, at least one of the exogenous genesencodes a fatty acyl-ACP thioesterase selected from the group consistingof the thioesterases identified in Table 4.

In a tenth aspect, the present invention provides a method of making anoil-based product. In one embodiment, the method comprises (a)cultivating a cell as discussed above until the cell is at least 5% orat least 15%, preferably at least 10%, lipid by dry weight, (b)separating the lipid from water-soluble biomass components, and (c)subjecting the lipid to at least one chemical reaction selected from thegroup consisting of: saponification; metathesis; acid hydrolysis;alkaline hydrolysis; enzymatic hydrolysis; catalytic hydrolysis;hot-compressed water hydrolysis; a catalytic hydrolysis reaction whereinthe lipid is split into glycerol and fatty acids; an amination reactionto produce fatty nitrogen compounds; an ozonolysis reaction to producemono- and dibasic-acids; a triglyceride splitting reaction selected fromthe group consisting of enzymatic splitting and pressure splitting; acondensation reaction that follows a hydrolysis reaction; ahydroprocessing reaction; a hydroprocessing reaction and a deoxygenationreaction or a condensation reaction prior to or simultaneous with thehydroprocessing reaction; a gas removal reaction; a deoxygenationreaction selected from the group consisting of a hydrogenolysisreaction, hydrogenation, a consecutive hydrogenation-hydrogenolysisreaction, a consecutive hydrogenolysis-hydrogenation reaction, and acombined hydrogenation-hydrogenolysis reaction; a condensation reactionfollowing a deoxygenation reaction; an esterification reaction; aninterestification reaction; a transesterification reaction; ahydroxylation reaction; and a condensation reaction following ahydroxylation reaction, whereby an oil-based product is produced.

In some cases, the oil-based product is selected from soap or a fuelproduct. In some embodiments, the oil-based product is a fuel productselected from the group consisting biodiesel, renewable diesel, and jetfuel. In some cases, the fuel product is biodiesel with one or more ofthe following attributes: (i) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g,preferably 0.05-0.244 mcg/g, total carotenoids; (ii) less than 0.01mcg/g, less than 0.005 mcg/g, preferably less than 0.003 mcg/g,lycopene; (iii) less than 0.01 mcg/g, less than 0.005 mcg/g, preferablyless than 0.003 mcg/g, beta carotene; (iv) 0.01-0.5 mcg/g, 0.025-0.3mcg/g, preferably 0.045-0.268 mcg/g, chlorophyll A; (v) 1-500 mcg/g,35-175 mcg/g, preferably 38.3-164 mcg/g, gamma tocopherol; (vi) lessthan 1%, less than 0.5%, preferably less than 0.25%, brassicasterol,campesterol, stignasterol, or beta-sitosterol; (vii) 100-500 mcg/g,225-350 mcg/g, preferably 249.6-325.3 mcg/g, total tocotrienols; (viii)0.001-0.1 mcg/g, 0.0025-0.05 mcg/g, preferably 0.003-0.039 mcg/g,lutein; or (ix) 10-500 mcg/g, 50-300 mcg/g, preferably 60.8-261.7 mcg/g,tocopherols. In some cases, the fuel product is renewable diesel thathas a T10-T90 of at least 20° C., 40° C. or 60° C. In some cases, thefuel product is jet fuel that meets HRJ-5 and/or ASTM specificationD1655.

In an eleventh aspect, the present invention provides a triglyceride oilcomprising (a) a lipid profile of at least 3% C8:0, at least 4% C10:0,at least 13% C12:0, at least 10% C14:0, and/or at least 60% saturatedfatty acids, and (b) one or more of the following attributes: (i)0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.05-0.244 mcg/g, totalcarotenoids; (ii) less than 0.01 mcg/g, less than 0.005 mcg/g,preferably less than 0.003 mcg/g, lycopene; (iii) less than 0.01 mcg/g,less than 0.005 mcg/g, preferably less than 0.003 mcg/g, beta carotene;(iv) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.045-0.268 mcg/g,chlorophyll A; (v) 1-300 mcg/g, 35-175 mcg/g, preferably 38.3-164 mcg/g,gamma tocopherol; (vi) less than 1%, less than 0.5%, preferably lessthan 0.25%, brassicasterol, campesterol, stignasterol, orbeta-sitosterol; (vii) 100-500 mcg/g, 225-350 mcg/g, preferably249.6-325.3 mcg/g, total tocotrienols; (viii) 0.001-0.1 mcg/g,0.0025-0.05 mcg/g, preferably 0.003-0.039 mcg/g, lutein; or (ix) 10-500mcg/g, 50-300 mcg/g, preferably 60.8-261.7 mcg/g, tocopherols.

In a twelfth aspect, the present invention provides an isolated oil frommicroalgae that has a C8:C10 fatty acid ratio of at least 5:1. In arelated aspect, the present invention provides an isolated oil frommicroalgae with at least 50% to 75%, preferably at least 60%, saturatedfatty acids. In another related aspect, the present invention providesan isolated oil from microalgae that has a C16:14 fatty acid ratio ofabout 2:1. In still another related aspect, the present inventionprovides an isolated oil from microalgae that has a C12:C14 fatty acidratio of at least 5:1. In some embodiments, the microalgae contains atleast one exogenous gene. In some cases, the microalgae is of the genusPrototheca.

In a thirteenth aspect, the present invention provides a triglycerideoil comprising (a) a lipid profile of less than 5% or less than 2%,preferably less than 1%, <C12; between 1%-10%, preferably 2%-7%, C14:0;between 20%-35%, preferably 23%-30%, C16:0; between 5%-20%, preferably7%-15%, C18:0; between 35-60%, preferably 40-55%, C18:1; and between1%-20%, preferably 2-15%, C18:2 fatty acids; and (b) one or more of thefollowing attributes: (i) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably0.05-0.244 mcg/g, total carotenoids; (ii) less than 0.01 mcg/g, lessthan 0.005 mcg/g, preferably less than 0.003 mcg/g, lycopene; (iii) lessthan 0.01 mcg/g, less than 0.005 mcg/g, preferably less than 0.003mcg/g, beta carotene; (iv) 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably0.045-0.268 mcg/g, chlorophyll A; (v) 1-300 mcg/g, 35-175 mcg/g,preferably 38.3-164 mcg/g, gamma tocopherol; (vi) less than 1%, lessthan 0.5%, preferably less than 0.25%, brassicasterol, campesterol,stignasterol, or beta-sitosterol; (vii) 100-500 mcg/g, 225-350 mcg/g,preferably 249.6-325.3 mcg/g, total tocotrienols; (viii) 0.001-0.1mcg/g, 0.0025-0.05 mcg/g, preferably 0.003-0.039 mcg/g, lutein; or (ix)10-500 mcg/g, 50-300, preferably 60.8-261.7 mcg/g, tocopherols.

In some cases, the triglyceride oil is isolated from a microbecomprising one or more exogenous gene. In some embodiments, the one ormore exogenous gene encodes a fatty acyl-ACP thioesterase. In somecases, the fatty acyl-ACP thioesterase has hydrolysis activity towardsfatty acyl-ACP substrates of chain length C14. In some embodiments, themicrobe further comprises a mutated endogenous desaturase gene, whereinthe mutation renders the gene or desaturase inactive.

In a fourteenth aspect, the present invention provides a method ofproducing a triglyceride oil comprising a lipid profile of less than 5%,or less than 2%, preferably less than 1%, <C12; between 1%-10%,preferably 2%-7%, C14:0; between 20%-35%, preferably 23%-30%, C16:0;between 5%-20%, preferably 7%-15%, C18:0; between 35%-60%, preferably40-55%, C18:1; and between 1%-20%, preferably 2-15%, C18:2 fatty acids,wherein the triglyceride oil is isolated from a microbe comprising oneor more exogenous gene. In some cases, the triglyceride oil comprises alipid profile of 1%-10%, preferably 3-5%, C14:0; 20%-30%, preferably25-27%, C16:0; 5%-20%, preferably 10-15%, C18:0; and 35%-50%, preferably40-45%, C18:1. In some embodiments, the one or more exogenous geneencodes a fatty acyl-ACP thioesterase. In some cases, the fatty acyl-ACPthioesterase has hydrolysis activity towards fatty acyl-ACP substratesof chain length C14. In some cases, the microbe further comprises amutated endogenous desaturase gene, wherein the mutation renders thegene or desaturase inactive. In some cases, the one or more exogenousgene is a sucrose invertase. In some embodiments, the mutated endogenousdesaturase gene is a stearoyl-acyl carrier protein desaturase (SAD)(e.g., SEQ ID NOs: 199-200). In some embodiments, the mutated endogenousdesaturase gene is a fatty acid desaturase (FAD).

In a fifteenth aspect, the present invention provides a oleaginousmicrobial cell, preferably a microalgal cell, comprising a triglycerideoil, wherein the fatty acid profile of the triglyceride oil is selectedfrom the group consisting of at least about 1% C8:0, at least about 1%C10:0, at least about 1% C12:0, at least about 2% C14:0, at least about30% C16:0, at least about 5% C18:0, at least about 60% C18:1, less thanabout 7% C18:2, and at least about 35% saturated fatty acids. In somecases, the oleaginous microbial cell comprises an exogenous gene, andoptionally, an endogenous desaturase of the oleaginous microbial cellhas been inactivated or mutated to have less enzymatic activity.

In some cases, the fatty acid profile of the triglyceride oil is similarto the fatty acid profile of a naturally occurring oil. In some cases,the naturally occurring oil is selected from the group consisting ofcocoa butter, coconut oil, palm oil, palm kernel oil, shea butter, beeftallow and lard. In some cases, the fatty acid profile of thetriglyceride oil comprises a profile selected from the group consistingof, the total combined amounts of C8:0 and C10:0 is at least about 10%,the total combined amount of C10:0, C12:0, and C14:0 is at least about50%, the total combined amount of C16:0, C18:0 and C18:1 is at leastabout 60%, the total combined amount of C18:0, C18:1 and C18:2 is atleast about 60%, the total combined amount of C14:0, C16:0, C18:0 andC18:1 is at least about 60%, and the total combined amount of C18:1 andC18:2 is less than about 30%. In some cases, the fatty acid profile ofthe triglyceride oil comprises a ratio of fatty acids selected from thegroup consisting of C8:0 to C10:0 ratio of at least about 5 to 1, C10:0to C12:0 ratio of at least about 6 to 1, C12:0 to C14:0 ratio of atleast about 5 to 1, C14:0 to C12:0 ratio of at least about 7:1, andC14:0 to C16:0 ratio of at least about 1 to 2.

In some cases, the endogenous desaturase is selected from the groupconsisting of stearoyl ACP desaturase and delta 12 fatty aciddesaturase. In some cases, the exogenous gene is selected from the groupconsisting of a gene encoding an acyl-ACP thioesterase. In some cases,the exogenous gene encodes an acyl-ACP thioesterase selected from thegroup consisting of those identified in Table 4. In some cases, theoleaginous microbial cell further comprises a gene encoding a sucroseinvertase.

In various embodiments, the oleaginous microbial cell is a cell of amicroalgal genus or species selected from Achnanthes orientalis,Agmenellum, Amphiprora hyaline, Amphora coffeiformis, Amphoracoffeiformis linea, Amphora coffeiformis punctata, Amphora coffeiformistaylori, Amphora coffeiformis tenuis, Amphora delicatissima, Amphoradelicatissima capitata, Amphora sp., Anabaena, Ankistrodesmus,Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp.,Botryococcus braunii, Botryococcus sudeticus, Carteria, Chaetocerosgracilis, Chaetoceros muelleri, Chaetoceros muelleri subsalsum,Chaetoceros sp., Chlorella anitrata, Chlorella Antarctica, Chlorellaaureoviridis, Chlorella candida, Chlorella capsulate, Chlorelladesiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca,Chlorella fusca var. vacuolate, Chlorella glucotropha, Chlorellainfusionum, Chlorella infusionum var. actophila, Chlorella infusionumvar. auxenophila, Chlorella kessleri, Chlorella lobophora (strain SAG37.88), Chlorella luteoviridis, Chlorella luteoviridis var.aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata,Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna,Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorellaprotothecoides (including any of UTEX strains 1806, 411, 264, 256, 255,250, 249, 31, 29, 25, and CCAP strains 211/17 and 211/8d), Chlorellaprotothecoides var. acidicola, Chlorella regularis, Chlorella regularisvar. minima, Chlorella regularis var. umbricata, Chlorella reisiglii,Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea,Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorellasp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii,Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia,Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis,Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f.tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorellaxanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorellavulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium,Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Cryptomonas sp.,Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliellasp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate,Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliellapeircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola,Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta,Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena,Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp.,Gloeothamnion sp., Hymenomonas sp., Isochrysis aff galbana, Isochrysisgalbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614),Monoraphidium minutum, Monoraphidium sp., Nannochloris sp.,Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata,Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp.,Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschiadissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschiainconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschiapusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis,Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva,Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoriasp., Oscillatoria subbrevis, Pascheria acidophila, Pavlova sp., Phagus,Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysisdentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora,Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii,Pyramimonas sp., Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus,Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp.,Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosiraweissflogii, and Viridiella fridericiana.

In some cases, the oleaginous microbial cell is a cell of the genusPrototheca. In some cases, the oleaginous microbial cell is a cell ofthe genus Prototheca moriformis.

In some cases, the oleaginous microbial cell is an oleaginous yeastcell. In some cases, the oleaginous microbial cell is an oleaginousbacterial cell.

In some cases, the naturally occurring oil is cocoa butter and theexogenous gene comprises a Carthamus tinctorus thioesterase gene. Insome cases, the naturally occurring oil is coconut oil. In some cases,the naturally occurring oil is palm oil and the exogenous gene comprisesa Elaeis guiniensis thioesterase gene, a Cuphea hookeriana thioesterasegene, a combination of a Cuphea hookeriana KAS IV gene and a Cupheawrightii FATB2 gene, or a construct designed to disrupt an endogenousKAS II gene. In some cases, the naturally occurring oil is palm kerneloil and the exogenous gene comprises a combination of a Cuphea wrightiiFATB2 gene and a construct designed to disrupt an endogenous SAD2B gene.In some cases, the naturally occurring oil is shea butter. In somecases, the naturally occurring oil is beef tallow. In some cases, thenaturally occurring oil is lard and the exogenous gene comprises acombination of U. californica thioesterase gene and a construct designedto disrupt an endogenous SAD2B gene, a combination of a Garciniamangostana thioesterase gene and a construct designed to disrupt anendogenous SAD2B gene, a Brassica napus thioesterase gene, or a Cupheahookeriana thioesterase gene.

In a sixteenth aspect, the present invention provides an oleaginousmicrobial triglyceride oil composition, wherein the fatty acid profileof the triglyceride oil is selected from the group consisting of atleast about 1% C8:0, at least about 1% C10:0, at least about 1% C12:0,at least about 2% C14:0, at least about 30% C16:0, at least about 5%C18:0, at least about 60% C18:1, less than about 7% C18:2, and at leastabout 35% saturated fatty acids. In various embodiments, thetriglyceride oil composition is produced by cultivating a population ofoleaginous microbial cells or recombinant oleaginous microbial cells ina culture medium, wherein the oleaginous microbial cells are asdescribed above, in particular those described above in connection withthe fifteenth aspect of the invention.

In some cases, the oleaginous microbial triglyceride oil compositionfurther comprises an attribute selected from the group consisting of:(i) less than 0.3 mcg/g total carotenoids; (ii) less than 0.005 mcg/glycopene; (iii) less than 0.005 mcg/g beta carotene; (iv) less than 0.3mcg/g chlorophyll A; (v) less than 175 mcg/g gamma tocopherol; (vi) lessthan 0.25% brassicasterol, campesterol, stignasterol, orbeta-sitosterol; (vii) less than 350 mcg/g total tocotrienols; (viii)less than 0.05 mcg/g lutein; or (ix) less than 275 mcg/g tocopherols.

In a seventeenth aspect, the present invention provides a method ofproducing an oleaginous microbial triglyceride oil composition having afatty acid profile selected from the group consisting of at least about1% C8:0, at least about 1% C10:0, at least about 1% C12:0, at leastabout 2% C14:0, at least about 30% C16:0, at least about 5% C18:0, atleast about 60% C18:1, less than about 7% C18:2, and at least about 35%saturated fatty acids, wherein the method comprises the steps of: (a)cultivating a population of oleaginous microbial cells in a culturemedium until at least 10% of the dry cell weight of the oleaginousmicrobial cells is triglyceride oil; and (b) isolating the triglycerideoil composition from the oleaginous microbial cells. In variousembodiments, the triglyceride oil composition is produced viacultivation of a population of oleaginous microbial cells or recombinantoleaginous microbial cells as described above, in particular thosedescribed above in connection with the fifteenth aspect of theinvention.

In an eighteenth aspect, the present invention provides a method ofmaking an oil-based product, wherein the method comprises the steps of:(a) subjecting the oleaginous microbial triglyceride oil composition, asdescribed above in connection with the sixteenth aspect of theinvention, to at least one chemical reaction selected from the groupconsisting of: saponification; metathesis; acid hydrolysis; alkalinehydrolysis; enzymatic hydrolysis; catalytic hydrolysis; hot-compressedwater hydrolysis; a catalytic hydrolysis reaction wherein the lipid issplit into glycerol and fatty acids; an amination reaction to producefatty nitrogen compounds; an ozonolysis reaction to produce mono- anddibasic-acids; a triglyceride splitting reaction selected from the groupconsisting of enzymatic splitting and pressure splitting; a condensationreaction that follows a hydrolysis reaction; a hydroprocessing reaction;a hydroprocessing reaction and a deoxygenation reaction or acondensation reaction prior to or simultaneous with the hydroprocessingreaction; a gas removal reaction; a deoxygenation reaction selected fromthe group consisting of a hydrogenolysis reaction, hydrogenation, aconsecutive hydrogenation-hydrogenolysis reaction, a consecutivehydrogenolysis-hydrogenation reaction, and a combinedhydrogenation-hydrogenolysis reaction; a condensation reaction followinga deoxygenation reaction; an esterification reaction; aninterestification reaction; a transesterification reaction; ahydroxylation reaction; and a condensation reaction following ahydroxylation reaction; and (b) isolating the product of the reactionfrom the other components.

In some cases, the oil-based product is selected from the groupconsisting of a soap, a fuel, a dielectric fluid, a hydraulic fluid, aplasticizer, a lubricant, a heat transfer fluid, and a metal workingfluid. In some cases, the oil-based product is a fuel product selectedfrom the group consisting of: (a) biodiesel; (b) renewable diesel; and(c) jet fuel.

In some cases, the fuel product is biodiesel with one or more of thefollowing attributes: (i) less than 0.3 mcg/g total carotenoids; (ii)less than 0.005 mcg/g lycopene; (iii) less than 0.005 mcg/g betacarotene; (iv) less than 0.3 mcg/g chlorophyll A; (v) less than 175mcg/g gamma tocopherol; (vi) less than 0.25% brassicasterol,campesterol, stignasterol, or beta-sitosterol; (vii) less than 350 mcg/gtotal tocotrienols; (viii) less than 0.05 mcg/g lutein; or (ix) lessthan 275 mcg/g tocopherols.

In some cases, the fuel product is renewable diesel that has a T10-T90of at least 20° C., 40° C. or 60° C.

In some cases, the fuel product is jet fuel that meets HRJ-5 and/or ASTMspecification D1655.

These and other aspects and embodiments of the invention are describedin the accompanying drawing, a brief description of which immediatelyfollows, the detailed description of the invention below, and areexemplified in the examples below. Any or all of the features discussedabove and throughout the application can be combined in variousembodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chromatogram of renewable diesel produced from Protothecatriglyceride oil.

DETAILED DESCRIPTION OF THE INVENTION

The present invention arises from the discovery that Prototheca andcertain related microorganisms have unexpectedly advantageous propertiesfor the production of oils, fuels, and other hydrocarbon or lipidcompositions economically and in large quantities, as well as from thediscovery of methods and reagents for genetically altering thesemicroorganisms to improve these properties. The oils produced by thesemicroorganisms can be used in the transportation fuel, oleochemical,and/or food and cosmetic industries, among other applications.Transesterification of lipids yields long-chain fatty acid esters usefulas biodiesel. Other enzymatic and chemical processes can be tailored toyield fatty acids, aldehydes, alcohols, alkanes, and alkenes. In someapplications, renewable diesel, jet fuel, or other hydrocarbon compoundsare produced. The present invention also provides methods of cultivatingmicroalgae for increased productivity and increased lipid yield, and/orfor more cost-effective production of the compositions described herein.

This detailed description of the invention is divided into sections forthe convenience of the reader. Section I provides definitions of termsused herein. Section II provides a description of culture conditionsuseful in the methods of the invention. Section III provides adescription of genetic engineering methods and materials. Section IVprovides a description of genetic engineering of microorganisms (e.g.,Prototheca) to enable sucrose utilization. Section V provides adescription of genetic engineering of microorganisms (e.g., Prototheca)to modify lipid biosynthesis. Section VI describes methods for makingfuels and chemicals. Section VII discloses examples and embodiments ofthe invention. The detailed description of the invention is followed byexamples that illustrate the various aspects and embodiments of theinvention.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

The following references provide one of skill with a general definitionof many of the terms used in this invention: Singleton et al.,Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); TheCambridge Dictionary of Science and Technology (Walker ed., 1988); TheGlossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag(1991); and Hale & Marham, The Harper Collins Dictionary of Biology(1991). As used herein, the following terms have the meanings ascribedto them unless specified otherwise.

“Active in microalgae” refers to a nucleic acid that is functional inmicroalgae. For example, a promoter that has been used to drive anantibiotic resistance gene to impart antibiotic resistance to atransgenic microalgae is active in microalgae.

“Acyl carrier protein” or “ACP” is a protein that binds a growing acylchain during fatty acid synthesis as a thiol ester at the distal thiolof the 4′-phosphopantetheine moiety and comprises a component of thefatty acid synthase complex.

“Acyl-CoA molecule” or “acyl-CoA” is a molecule comprising an acylmoiety covalently attached to coenzyme A through a thiol ester linkageat the distal thiol of the 4′-phosphopantetheine moiety of coenzyme A.

“Area Percent” refers to the area of peaks observed using FAME GC/FIDdetection methods in which every fatty acid in the sample is convertedinto a fatty acid methyl ester (FAME) prior to detection. For example, aseparate peak is observed for a fatty acid of 14 carbon atoms with nounsaturation (C14:0) compared to any other fatty acid such as C14:1. Thepeak area for each class of FAME is directly proportional to its percentcomposition in the mixture and is calculated based on the sum of allpeaks present in the sample (i.e. [area under specific peak/total areaof all measured peaks]×100). When referring to lipid profiles of oilsand cells of the invention, “at least 4% C8-C14” means that at least 4%of the total fatty acids in the cell or in the extracted glycerolipidcomposition have a chain length that includes 8, 10, 12 or 14 carbonatoms.

“Axenic” is a culture of an organism free from contamination by otherliving organisms.

“Biodiesel” is a biologically produced fatty acid alkyl ester suitablefor use as a fuel in a diesel engine.

“Biomass” is material produced by growth and/or propagation of cells.Biomass may contain cells and/or intracellular contents as well asextracellular material, includes, but is not limited to, compoundssecreted by a cell.

“Bioreactor” is an enclosure or partial enclosure in which cells arecultured, optionally in suspension.

“Catalyst” is an agent, such as a molecule or macromolecular complex,capable of facilitating or promoting a chemical reaction of a reactantto a product without becoming a part of the product. A catalystincreases the rate of a reaction, after which, the catalyst may act onanother reactant to form the product. A catalyst generally lowers theoverall activation energy required for the reaction such that itproceeds more quickly or at a lower temperature. Thus, a reactionequilibrium may be more quickly attained. Examples of catalysts includeenzymes, which are biological catalysts; heat, which is a non-biologicalcatalyst; and metals used in fossil oil refining processes.

“Cellulosic material” is the product of digestion of cellulose,including glucose and xylose, and optionally additional compounds suchas disaccharides, oligosaccharides, lignin, furfurals and othercompounds. Nonlimiting examples of sources of cellulosic materialinclude sugar cane bagasses, sugar beet pulp, corn stover, wood chips,sawdust and switchgrass.

“Co-culture”, and variants thereof such as “co-cultivate” and“co-ferment”, refer to the presence of two or more types of cells in thesame bioreactor. The two or more types of cells may both bemicroorganisms, such as microalgae, or may be a microalgal cell culturedwith a different cell type. The culture conditions may be those thatfoster growth and/or propagation of the two or more cell types or thosethat facilitate growth and/or proliferation of one, or a subset, of thetwo or more cells while maintaining cellular growth for the remainder.

“Cofactor” is any molecule, other than the substrate, required for anenzyme to carry out its enzymatic activity.

“Complementary DNA” or “cDNA” is a DNA copy of mRNA, usually obtained byreverse transcription of messenger RNA (mRNA) or amplification (e.g.,via polymerase chain reaction (“PCR”)).

“Cultivated”, and variants thereof such as “cultured” and “fermented”,refer to the intentional fostering of growth (increases in cell size,cellular contents, and/or cellular activity) and/or propagation(increases in cell numbers via mitosis) of one or more cells by use ofselected and/or controlled conditions. The combination of both growthand propagation may be termed proliferation. Examples of selected and/orcontrolled conditions include the use of a defined medium (with knowncharacteristics such as pH, ionic strength, and carbon source),specified temperature, oxygen tension, carbon dioxide levels, and growthin a bioreactor. Cultivate does not refer to the growth or propagationof microorganisms in nature or otherwise without human intervention; forexample, natural growth of an organism that ultimately becomesfossilized to produce geological crude oil is not cultivation.

“Cytolysis” is the lysis of cells in a hypotonic environment. Cytolysisis caused by excessive osmosis, or movement of water, towards the insideof a cell (hyperhydration). The cell cannot withstand the osmoticpressure of the water inside, and so it explodes.

“Delipidated meal” and “delipidated microbial biomass” is microbialbiomass after oil (including lipids) has been extracted or isolated fromit, either through the use of mechanical (i.e., exerted by an expellerpress) or solvent extraction or both. Delipidated meal has a reducedamount of oil/lipids as compared to before the extraction or isolationof oil/lipids from the microbial biomass but does contain some residualoil/lipid.

“Expression vector” or “expression construct” or “plasmid” or“recombinant DNA construct” refer to a nucleic acid that has beengenerated via human intervention, including by recombinant means ordirect chemical synthesis, with a series of specified nucleic acidelements that permit transcription and/or translation of a particularnucleic acid in a host cell. The expression vector can be part of aplasmid, virus, or nucleic acid fragment. Typically, the expressionvector includes a nucleic acid to be transcribed operably linked to apromoter.

“Exogenous gene” is a nucleic acid that codes for the expression of anRNA and/or protein that has been introduced (“transformed”) into a cell.A transformed cell may be referred to as a recombinant cell, into whichadditional exogenous gene(s) may be introduced. The exogenous gene maybe from a different species (and so heterologous), or from the samespecies (and so homologous), relative to the cell being transformed.Thus, an exogenous gene can include a homologous gene that occupies adifferent location in the genome of the cell or is under differentcontrol, relative to the endogenous copy of the gene. An exogenous genemay be present in more than one copy in the cell. An exogenous gene maybe maintained in a cell as an insertion into the genome or as anepisomal molecule.

“Exogenously provided” refers to a molecule provided to the culturemedia of a cell culture.

“Expeller pressing” is a mechanical method for extracting oil from rawmaterials such as soybeans and rapeseed. An expeller press is a screwtype machine, which presses material through a caged barrel-like cavity.Raw materials enter one side of the press and spent cake exits the otherside while oil seeps out between the bars in the cage and is collected.The machine uses friction and continuous pressure from the screw drivesto move and compress the raw material. The oil seeps through smallopenings that do not allow solids to pass through. As the raw materialis pressed, friction typically causes it to heat up.

“Fatty acyl-ACP thioesterase” is an enzyme that catalyzes the cleavageof a fatty acid from an acyl carrier protein (ACP) during lipidsynthesis.

“Fatty acyl-CoA/aldehyde reductase” is an enzyme that catalyzes thereduction of an acyl-CoA molecule to a primary alcohol.

“Fatty acyl-CoA reductase” is an enzyme that catalyzes the reduction ofan acyl-CoA molecule to an aldehyde.

“Fatty aldehyde decarbonylase” is an enzyme that catalyzes theconversion of a fatty aldehyde to an alkane.

“Fatty aldehyde reductase” is an enzyme that catalyzes the reduction ofan aldehyde to a primary alcohol.

“Fixed carbon source” is a molecule(s) containing carbon, typically anorganic molecule, that is present at ambient temperature and pressure insolid or liquid form in a culture media that can be utilized by amicroorganism cultured therein.

“Homogenate” is biomass that has been physically disrupted.

“Hydrocarbon” is (a) a molecule containing only hydrogen and carbonatoms wherein the carbon atoms are covalently linked to form a linear,branched, cyclic, or partially cyclic backbone to which the hydrogenatoms are attached. The molecular structure of hydrocarbon compoundsvaries from the simplest, in the form of methane (CH₄), which is aconstituent of natural gas, to the very heavy and very complex, such assome molecules such as asphaltenes found in crude oil, petroleum, andbitumens. Hydrocarbons may be in gaseous, liquid, or solid form, or anycombination of these forms, and may have one or more double or triplebonds between adjacent carbon atoms in the backbone. Accordingly, theterm includes linear, branched, cyclic, or partially cyclic alkanes,alkenes, lipids, and paraffin. Examples include propane, butane,pentane, hexane, octane, and squalene.

“Hydrogen:carbon ratio” is the ratio of hydrogen atoms to carbon atomsin a molecule on an atom-to-atom basis. The ratio may be used to referto the number of carbon and hydrogen atoms in a hydrocarbon molecule.For example, the hydrocarbon with the highest ratio is methane CH₄(4:1).

“Hydrophobic fraction” is the portion, or fraction, of a material thatis more soluble in a hydrophobic phase in comparison to an aqueousphase. A hydrophobic fraction is substantially insoluble in water andusually non-polar.

“Increase lipid yield” refers to an increase in the productivity of amicrobial culture by, for example, increasing dry weight of cells perliter of culture, increasing the percentage of cells that constitutelipid, or increasing the overall amount of lipid per liter of culturevolume per unit time.

“Inducible promoter” is a promoter that mediates transcription of anoperably linked gene in response to a particular stimulus. Examples ofsuch promoters may be promoter sequences that are induced in conditionsof changing pH or nitrogen levels.

“In operable linkage” is a functional linkage between two nucleic acidsequences, such a control sequence (typically a promoter) and the linkedsequence (typically a sequence that encodes a protein, also called acoding sequence). A promoter is in operable linkage with an exogenousgene if it can mediate transcription of the gene.

“In situ” means “in place” or “in its original position”.

“Limiting concentration of a nutrient” is a concentration of a compoundin a culture that limits the propagation of a cultured organism. A“non-limiting concentration of a nutrient” is a concentration thatsupports maximal propagation during a given culture period. Thus, thenumber of cells produced during a given culture period is lower in thepresence of a limiting concentration of a nutrient than when thenutrient is non-limiting. A nutrient is said to be “in excess” in aculture, when the nutrient is present at a concentration greater thanthat which supports maximal propagation.

“Lipase” is a water-soluble enzyme that catalyzes the hydrolysis ofester bonds in water-insoluble, lipid substrates. Lipases catalyze thehydrolysis of lipids into glycerols and fatty acids.

“Lipid modification enzyme” refers to an enayme that alters the covalentstructure of a lipid. Examples of lipid modification enzymes include alipase, a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehydereductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, adesaturase, including a stearoyl acyl carrier protein desaturase (SAD)and a fatty acyl destaurase (FAD), and a fatty aldehyde decarbonylase.

“Lipid pathway enzyme” is any enzyme that plays a role in lipidmetabolism, i.e., either lipid synthesis, modification, or degradation,and any proteins that chemically modify lipids, as well as carrierproteins.

“Lipids” are a class of molecules that are soluble in nonpolar solvents(such as ether and chloroform) and are relatively or completelyinsoluble in water. Lipid molecules have these properties, because theyconsist largely of long hydrocarbon tails which are hydrophobic innature. Examples of lipids include fatty acids (saturated andunsaturated); glycerides or glycerolipids (such as monoglycerides,diglycerides, triglycerides or neutral fats, and phosphoglycerides orglycerophospholipids); nonglycerides (sphingolipids, sterol lipidsincluding cholesterol and steroid hormones, prenol lipids includingterpenoids, fatty alcohols, waxes, and polyketides); and complex lipidderivatives (sugar-linked lipids, or glycolipids, and protein-linkedlipids). “Fats” are a subgroup of lipids called “triacylglycerides.”

“Lysate” is a solution containing the contents of lysed cells.

“Lysis” is the breakage of the plasma membrane and optionally the cellwall of a biological organism sufficient to release at least someintracellular content, often by mechanical, viral or osmotic mechanismsthat compromise its integrity.

“Lysing” is disrupting the cellular membrane and optionally the cellwall of a biological organism or cell sufficient to release at leastsome intracellular content.

“Microalgae” is a eukarytotic microbial organism that contains achloroplast or plastid, and optionally that is capable of performingphotosynthesis, or a prokaryotic microbial organism capable ofperforming photosynthesis. Microalgae include obligate photoautotrophs,which cannot metabolize a fixed carbon source as energy, as well asheterotrophs, which can live solely off of a fixed carbon source.Microalgae include unicellular organisms that separate from sister cellsshortly after cell division, such as Chlamydomonas, as well as microbessuch as, for example, Volvox, which is a simple multicellularphotosynthetic microbe of two distinct cell types. Microalgae includecells such as Chlorella, Dunaliella, and Prototheca. Microalgae alsoinclude other microbial photosynthetic organisms that exhibit cell-celladhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae alsoinclude obligate heterotrophic microorganisms that have lost the abilityto perform photosynthesis, such as certain dinoflagellate algae speciesand species of the genus Prototheca.

“Microorganism” and “microbe” are microscopic unicellular organisms.

“Naturally co-expressed” with reference to two proteins or genes meansthat the proteins or their genes are co-expressed naturally in a tissueor organism from which they are derived, e.g., because the genesencoding the two proteins are under the control of a common regulatorysequence or because they are expressed in response to the same stimulus.

“Osmotic shock” is the rupture of cells in a solution following a suddenreduction in osmotic pressure. Osmotic shock is sometimes induced torelease cellular components of such cells into a solution.

“Polysaccharide-degrading enzyme” is any enzyme capable of catalyzingthe hydrolysis, or saccharification, of any polysaccharide. For example,cellulases catalyze the hydrolysis of cellulose.

“Polysaccharides” or “glycans” are carbohydrates made up ofmonosaccharides joined together by glycosidic linkages. Cellulose is apolysaccharide that makes up certain plant cell walls. Cellulose can bedepolymerized by enzymes to yield monosaccharides such as xylose andglucose, as well as larger disaccharides and oligosaccharides.

“Promoter” is a nucleic acid control sequence that directs transcriptionof a nucleic acid. As used herein, a promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements, which can belocated as much as several thousand base pairs from the start site oftranscription.

“Recombinant” is a cell, nucleic acid, protein or vector, that has beenmodified due to the introduction of an exogenous nucleic acid or thealteration of a native nucleic acid. Thus, e.g., recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell or express native genes differently than those genesare expressed by a non-recombinant cell. A “recombinant nucleic acid” isa nucleic acid originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,or otherwise is in a form not normally found in nature. Recombinantnucleic acids may be produced, for example, to place two or more nucleicacids in operable linkage. Thus, an isolated nucleic acid or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined in nature, are both considered recombinant for thepurposes of this invention. Once a recombinant nucleic acid is made andintroduced into a host cell or organism, it may replicate using the invivo cellular machinery of the host cell; however, such nucleic acids,once produced recombinantly, although subsequently replicatedintracellularly, are still considered recombinant for purposes of thisinvention. Similarly, a “recombinant protein” is a protein made usingrecombinant techniques, i.e., through the expression of a recombinantnucleic acid.

“Renewable diesel” is a mixture of alkanes (such as C10:0, C12:0, C14:0,C16:0 and C18:0) produced through hydrogenation and deoxygenation oflipids.

“Saccharification” is a process of converting biomass, usuallycellulosic or lignocellulosic biomass, into monomeric sugars, such asglucose and xylose. “Saccharified” or “depolymerized” cellulosicmaterial or biomass refers to cellulosic material or biomass that hasbeen converted into monomeric sugars through saccharification.

The term “similar,” when used in the context of a comparison to anaturally occurring oil, without further qualification, means that theoil being compared to the naturally occurring oil contains about +/−15%,or +/−10% of the top two triglycerides of the naturally occurring oil.For example, Shea butter (the oil of B. Parkii) contains 41.2-56.8%C18:0 and 34.0-46.9% C18:1 as the two most common triglyceridecomponents (see Table 5). A “similar” oil that is within +/−10% wouldcontain from about 37% to about 62% C18:0 and from 31% to about 52%C18:1 as the two most common triglyceride components. When used in thiscontext, the term “similar” includes +/−9%, +/−8%, +/−7%, +/−6%, +/−5%,+/−4%, +/−3%, +/−2%, or +/−1%, and can further represent a comparison tothe top three or top four triglycerides of the naturally occurring oil,or two out of the top three triglycerides, or three out of the top fourtriglycerides.

“Sonication” is a process of disrupting biological materials, such as acell, by use of sound wave energy.

“Species of furfural” is 2-furancarboxaldehyde or a derivative thatretains the same basic structural characteristics.

“Stover” is the dried stalks and leaves of a crop remaining after agrain has been harvested.

“Sucrose utilization gene” is a gene that, when expressed, aids theability of a cell to utilize sucrose as an energy source. Proteinsencoded by a sucrose utilization gene are referred to herein as “sucroseutilization enzymes” and include sucrose transporters, sucroseinvertases, and hexokinases such as glucokinases and fructokinases.

II. Cultivation

The present invention generally relates to cultivation of microorganisms(e.g., microalgae, oleaginous yeast, fungi, and bacteria), particularlyrecombinant microalgal strains, including Prototheca strains, for theproduction of lipid. For the convenience of the reader, this section issubdivided into subsections. Subsection 1 describes Prototheca speciesand strains and how to identify new Prototheca species and strains andrelated microalgae by genomic DNA comparison, as well as othermicroorganisms. Subsection 2 describes bioreactors useful forcultivation. Subsection 3 describes media for cultivation. Subsection 4describes oil production in accordance with illustrative cultivationmethods of the invention. These descriptions are also more generallyapplicable to other microorganisms.

1. Prototheca Species and Strains and Other Microorganisms

Prototheca is a remarkable microorganism for use in the production oflipid, because it can produce high levels of lipid, particularly lipidsuitable for fuel production. The lipid produced by Prototheca hashydrocarbon chains of shorter chain length and a higher degree ofsaturation than that produced by other microalgae. Moreover, Protothecalipid is generally free of pigment (low to undetectable levels ofchlorophyll and certain carotenoids) and in any event contains much lesspigment than lipid from other microalgae. Moreover, recombinantPrototheca cells provided by the invention can be used to produce lipidin greater yield and efficiency, and with reduced cost, relative to theproduction of lipid from other microorganisms. Illustrative Protothecastrains for use in the methods of the invention include In addition,this microalgae grows heterotrophically and can be geneticallyengineered as Prototheca wickerhamii, Prototheca stagnora (includingUTEX 327), Prototheca portoricensis, Prototheca moriformis (includingUTEX strains 1441, 1435), and Prototheca zopfii. Species of the genusPrototheca are obligate heterotrophs.

Species of Prototheca for use in the invention can be identified byamplification of certain target regions of the genome. For example,identification of a specific Prototheca species or strain can beachieved through amplification and sequencing of nuclear and/orchloroplast DNA using primers and methodology using any region of thegenome, for example using the methods described in Wu et al., Bot. Bull.Acad. Sin. (2001) 42:115-121 Identification of Chlorella spp. isolatesusing ribosomal DNA sequences. Well established methods of phylogeneticanalysis, such as amplification and sequencing of ribosomal internaltranscribed spacer (ITS1 and ITS2 rDNA), 23S rRNA, 18S rRNA, and otherconserved genomic regions can be used by those skilled in the art toidentify species of not only Prototheca, but other hydrocarbon and lipidproducing organisms with similar lipid profiles and productioncapability. For examples of methods of identification and classificationof algae also see for example Genetics, 2005 August; 170(4):1601-10 andRNA, 2005 April; 11(4):361-4.

Thus, genomic DNA comparison can be used to identify suitable species ofmicroalgae to be used in the present invention. Regions of conservedgenomic DNA, such as but not limited to DNA encoding for 23S rRNA, canbe amplified from microalgal species and compared to consensus sequencesin order to screen for microalgal species that are taxonomically relatedto the preferred microalgae used in the present invention. Examples ofsuch DNA sequence comparison for species within the Prototheca genus areshown below. Genomic DNA comparison can also be useful to identifymicroalgal species that have been misidentified in a strain collection.Often a strain collection will identify species of microalgae based onphenotypic and morphological characteristics. The use of thesecharacteristics may lead to miscategorization of the species or thegenus of a microalgae. The use of genomic DNA comparison can be a bettermethod of categorizing microalgae species based on their phylogeneticrelationship.

Microalgae for use in the present invention typically have genomic DNAsequences encoding for 23S rRNA that have at least 99%, least 95%, atleast 90%, or at least 85% nucleotide identity to at least one of thesequences listed in SEQ ID NOs: 11-19.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (at the web addresswww.ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra.). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. For identifying whether a nucleicacid or polypeptide is within the scope of the invention, the defaultparameters of the BLAST programs are suitable. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. TheTBLATN program (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix. (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Other considerations affecting the selection of microorganisms for usein the invention include, in addition to production of suitable lipidsor hydrocarbons for production of oils, fuels, and oleochemicals: (1)high lipid content as a percentage of cell weight; (2) ease of growth;(3) ease of genetic engineering; and (4) ease of biomass processing. Inparticular embodiments, the wild-type or genetically engineeredmicroorganism yields cells that are at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, or at least 70% or morelipid. Preferred organisms grow heterotrophically (on sugars in theabsence of light).

Examples of algae that can be used to practice the present inventioninclude, but are not limited to the following algae listed in Table 1.

TABLE 1 Examples of algae. Achnanthes orientalis, Agmenellum, Amphiprorahyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphoracoffeiformis punctata, Amphora coffeiformis taylori, Amphoracoffeiformis tenuis, Amphora delicatissima, Amphora delicatissimacapitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmusfalcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii,Botryococcus sudeticus, Carteria, Chaetoceros gracilis, Chaetocerosmuelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorellaanitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorellacandida, Chlorella capsulate, Chlorella desiccate, Chlorellaellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var.vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorellainfusionum var. actophila, Chlorella infusionum var. auxenophila,Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorellaluteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorellaluteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima,Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorellaphotophila, Chlorella pringsheimii, Chlorella protothecoides (includingany of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25, andCCAP strains 211/17 and 211/8d), Chlorella protothecoides var.acidicola, Chlorella regularis, Chlorella regularis var. minima,Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorellasaccharophila, Chlorella saccharophila var. ellipsoidea, Chlorellasalina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp.,Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii,Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia,Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis,Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f.tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorellaxanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorellavulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium,Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Cryptomonas sp.,Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliellasp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate,Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliellapeircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola,Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta,Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena,Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp.,Gloeothamnion sp., Hymenomonas sp., Isochrysis aff. galbana, Isochrysisgalbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614),Monoraphidium minutum, Monoraphidium sp., Nannochloris sp.,Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata,Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp.,Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschiadissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschiainconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschiapusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis,Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva,Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoriasp., Oscillatoria subbrevis, Pascheria acidophila, Pavlova sp., Phagus,Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysisdentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora,Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii,Pyramimonas sp., Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus,Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp.,Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosiraweissflogii, and Viridiella fridericiana

Examples of oleaginous yeast that can be used to practice the presentinvention include, but are not limited to the following oleaginous yeastlisted in Table 1A.

Table 1A. Examples of oleaginous yeast.

Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomycesstarkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorulaglutinis, Rhodotorula gracilis, and Yarrowia lipolytica

Examples of other fungi that can be used to practice the presentinvention include, but are not limited to the following fungi listed inTable 1B.

Table 1B. Examples of fungi.

Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum,Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus,Pennicillium iilacinum, Hensenulo, Chaetomium, Cladosporium,Malbranchea, Rhizopus, and Pythium

In some embodiments of the present invention, the microorganism is abacterium. Examples of expression of exogenous genes in bacteria, suchas E. coli, are well known; see for example Molecular Cloning: ALaboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring HarborPress).

2. Bioreactor

Microrganisms are cultured both for purposes of conducting geneticmanipulations and for production of hydrocarbons (e.g., lipids, fattyacids, aldehydes, alcohols, and alkanes). The former type of culture isconducted on a small scale and initially, at least, under conditions inwhich the starting microorganism can grow. Culture for purposes ofhydrocarbon production is usually conducted on a large scale (e.g.,10,000 L, 40,000 L, 100,000 L or larger bioreactors) in a bioreactor.Microalgae, including Prototheca species are typically cultured in themethods of the invention in liquid media within a bioreactor. Typically,the bioreactor does not allow light to enter.

The bioreactor or fermentor is used to culture oleaginous microbialcells, preferably microalgal cells through the various phases of theirphysiological cycle. Bioreactors offer many advantages for use inheterotrophic growth and propagation methods. To produce biomass for usein food, microalgae are preferably fermented in large quantities inliquid, such as in suspension cultures as an example. Bioreactors suchas steel fermentors can accommodate very large culture volumes (40,000liter and greater capacity bioreactors are used in various embodimentsof the invention). Bioreactors also typically allow for the control ofculture conditions such as temperature, pH, oxygen tension, and carbondioxide levels. For example, bioreactors are typically configurable, forexample, using ports attached to tubing, to allow gaseous components,like oxygen or nitrogen, to be bubbled through a liquid culture. Otherculture parameters, such as the pH of the culture media, the identityand concentration of trace elements, and other media constituents canalso be more readily manipulated using a bioreactor.

Bioreactors can be configured to flow culture media though thebioreactor throughout the time period during which the microalgaereproduce and increase in number. In some embodiments, for example,media can be infused into the bioreactor after inoculation but beforethe cells reach a desired density. In other instances, a bioreactor isfilled with culture media at the beginning of a culture, and no moreculture media is infused after the culture is inoculated. In otherwords, the microalgal biomass is cultured in an aqueous medium for aperiod of time during which the microalgae reproduce and increase innumber; however, quantities of aqueous culture medium are not flowedthrough the bioreactor throughout the time period. Thus in someembodiments, aqueous culture medium is not flowed through the bioreactorafter inoculation.

Bioreactors equipped with devices such as spinning blades and impellers,rocking mechanisms, stir bars, means for pressurized gas infusion can beused to subject microalgal cultures to mixing. Mixing may be continuousor intermittent. For example, in some embodiments, a turbulent flowregime of gas entry and media entry is not maintained for reproductionof microalgae until a desired increase in number of said microalgae hasbeen achieved.

Bioreactor ports can be used to introduce, or extract, gases, solids,semisolids, and liquids, into the bioreactor chamber containing themicroalgae. While many bioreactors have more than one port (for example,one for media entry, and another for sampling), it is not necessary thatonly one substance enter or leave a port. For example, a port can beused to flow culture media into the bioreactor and later used forsampling, gas entry, gas exit, or other purposes. Preferably, a samplingport can be used repeatedly without altering compromising the axenicnature of the culture. A sampling port can be configured with a valve orother device that allows the flow of sample to be stopped and started orto provide a means of continuous sampling. Bioreactors typically have atleast one port that allows inoculation of a culture, and such a port canalso be used for other purposes such as media or gas entry.

Bioreactors ports allow the gas content of the culture of microalgae tobe manipulated. To illustrate, part of the volume of a bioreactor can begas rather than liquid, and the gas inlets of the bioreactor to allowpumping of gases into the bioreactor. Gases that can be beneficiallypumped into a bioreactor include air, air/CO₂ mixtures, noble gases,such as argon, and other gases. Bioreactors are typically equipped toenable the user to control the rate of entry of a gas into thebioreactor. As noted above, increasing gas flow into a bioreactor can beused to increase mixing of the culture.

Increased gas flow affects the turbidity of the culture as well.Turbulence can be achieved by placing a gas entry port below the levelof the aqueous culture media so that gas entering the bioreactor bubblesto the surface of the culture. One or more gas exit ports allow gas toescape, thereby preventing pressure buildup in the bioreactor.Preferably a gas exit port leads to a “one-way” valve that preventscontaminating microorganisms from entering the bioreactor.

3. Media

Microalgal culture media typically contains components such as a fixednitrogen source, a fixed carbon source, trace elements, optionally abuffer for pH maintenance, and phosphate (typically provided as aphosphate salt). Other components can include salts such as sodiumchloride, particularly for seawater microalgae. Nitrogen sources includeorganic and inorganic nitrogen sources, including, for example, withoutlimitation, molecular nitrogen, nitrate, nitrate salts, ammonia (pure orin salt form, such as, (NH₄)₂SO₄ and NH₄OH), protein, soybean meal,cornsteep liquor, and yeast extract. Examples of trace elements includezinc, boron, cobalt, copper, manganese, and molybdenum in, for example,the respective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂Oand (NH₄)₆Mo₇O₂₄. 4H₂O.

Microorganisms useful in accordance with the methods of the presentinvention are found in various locations and environments throughout theworld. As a consequence of their isolation from other species and theirresulting evolutionary divergence, the particular growth medium foroptimal growth and generation of lipid and/or hydrocarbon constituentscan be difficult to predict. In some cases, certain strains ofmicroorganisms may be unable to grow on a particular growth mediumbecause of the presence of some inhibitory component or the absence ofsome essential nutritional requirement required by the particular strainof microorganism.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismscan be found, for example, online at www.utex.org/, a site maintained bythe University of Texas at Austin, 1 University Station A6700, Austin,Tex., 78712-0183, for its culture collection of algae (UTEX). Forexample, various fresh water and salt water media include thosedescribed in PCT Pub. No. 2008/151149, incorporated herein by reference.

In a particular example, Proteose Medium is suitable for axeniccultures, and a 1 L volume of the medium (pH ˜6.8) can be prepared byaddition of 1 g of proteose peptone to 1 liter of Bristol Medium.Bristol medium comprises 2.94 mM NaNO₃, 0.17 mM CaCl₂.2H₂O, 0.3 mMMgSO₄.7H₂O, 0.43 mM, 1.29 mM KH₂PO₄, and 1.43 mM NaCl in an aqueoussolution. For 1.5% agar medium, 15 g of agar can be added to 1 L of thesolution. The solution is covered and autoclaved, and then stored at arefrigerated temperature prior to use. Another example is the Protothecaisolation medium (PIM), which comprises 10 g/L postassium hydrogenphthalate (KHP), 0.9 g/L sodium hydroxide, 0.1 g/L magnesium sulfate,0.2 g/L potassium hydrogen phosphate, 0.3 g/L ammonium chloride, 10 g/Lglucose 0.001 g/L thiamine hydrochloride, 20 g/L agar, 0.25 g/L5-fluorocytosine, at a pH in the range of 5.0 to 5.2 (see Pore, 1973,App. Microbiology, 26: 648-649). Other suitable media for use with themethods of the invention can be readily identified by consulting the URLidentified above, or by consulting other organizations that maintaincultures of microorganisms, such as SAG, CCAP, or CCALA. SAG refers tothe Culture Collection of Algae at the University of Gottingen(Gottingen, Germany), CCAP refers to the culture collection of algae andprotozoa managed by the Scottish Association for Marine Science(Scotland, United Kingdom), and CCALA refers to the culture collectionof algal laboratory at the Institute of Botany (T{hacek over(r)}ebo{hacek over (n)}, Czech Republic). Additionally, U.S. Pat. No.5,900,370 describes media formulations and conditions suitable forheterotrophic fermentation of Prototheca species.

For oil production, selection of a fixed carbon source is important, asthe cost of the fixed carbon source must be sufficiently low to make oilproduction economical. Thus, while suitable carbon sources include, forexample, acetate, floridoside, fructose, galactose, glucuronic acid,glucose, glycerol, lactose, mannose, N-acetylglucosamine, rhamnose,sucrose, and/or xylose, selection of feedstocks containing thosecompounds is an important aspect of the methods of the invention.Suitable feedstocks useful in accordance with the methods of theinvention include, for example, black liquor, corn starch, depolymerizedcellulosic material, milk whey, molasses, potato, sorghum, sucrose,sugar beet, sugar cane, rice, and wheat. Carbon sources can also beprovided as a mixture, such as a mixture of sucrose and depolymerizedsugar beet pulp. The one or more carbon source(s) can be supplied at aconcentration of at least about 50 μM, at least about 100 μM, at leastabout 500 μM, at least about 5 mM, at least about 50 mM, and at leastabout 500 mM, of one or more exogenously provided fixed carbonsource(s). Carbon sources of particular interest for purposes of thepresent invention include cellulose (in a depolymerized form), glycerol,sucrose, and sorghum, each of which is discussed in more detal below.

In accordance with the present invention, microorganisms can be culturedusing depolymerized cellulosic biomass as a feedstock. Cellulosicbiomass (e.g., stover, such as corn stover) is inexpensive and readilyavailable; however, attempts to use this material as a feedstock foryeast have failed. In particular, such feedstocks have been found to beinhibitory to yeast growth, and yeast cannot use the 5-carbon sugarsproduced from cellulosic materials (e.g., xylose from hemi-cellulose).By contrast, microalgae can grow on processed cellulosic material.Cellulosic materials generally include about 40-60% cellulose; about20-40% hemicellulose; and 10-30% lignin.

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves, not removed from the fields with theprimary food or fiber product. Examples include agricultural wastes suchas sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves,husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp,citrus peels; forestry wastes such as hardwood and softwood thinnings,and hardwood and softwood residues from timber operations; wood wastessuch as saw mill wastes (wood chips, sawdust) and pulp mill waste; urbanwastes such as paper fractions of municipal solid waste, urban woodwaste and urban green waste such as municipal grass clippings; and woodconstruction waste. Additional cellulosics include dedicated cellulosiccrops such as switchgrass, hybrid poplar wood, and miscanthus, fibercane, and fiber sorghum. Five-carbon sugars that are produced from suchmaterials include xylose.

Cellulosic materials are treated to increase the efficiency with whichthe microbe can utilize the sugar(s) contained within the materials. Theinvention provides novel methods for the treatment of cellulosicmaterials after acid explosion so that the materials are suitable foruse in a heterotrophic culture of microbes (e.g., microalgae andoleaginous yeast). As discussed above, lignocellulosic biomass iscomprised of various fractions, including cellulose, a crystallinepolymer of beta 1,4 linked glucose (a six-carbon sugar), hemicellulose,a more loosely associated polymer predominantly comprised of xylose (afive-carbon sugar) and to a lesser extent mannose, galactose, arabinose,lignin, a complex aromatic polymer comprised of sinapyl alcohol and itsderivatives, and pectins, which are linear chains of an alpha 1,4 linkedpolygalacturonic acid. Because of the polymeric structure of celluloseand hemicellulose, the sugars (e.g., monomeric glucose and xylose) inthem are not in a form that can be efficiently used (metabolized) bymany microbes. For such microbes, further processing of the cellulosicbiomass to generate the monomeric sugars that make up the polymers canbe very helpful to ensuring that the cellulosic materials areefficiently utilized as a feedstock (carbon source).

Celluose or cellulosic biomass is subjected to a process, termed“explosion”, in which the biomass is treated with dilute sulfuric (orother) acid at elevated temperature and pressure. This processconditions the biomass such that it can be efficiently subjected toenzymatic hydrolysis of the cellulosic and hemicellulosic fractions intoglucose and xylose monomers. The resulting monomeric sugars are termedcellulosic sugars. Cellulosic sugars can subsequently be utilized bymicroorganisms to produce a variety of metabolites (e.g., lipid). Theacid explosion step results in a partial hydrolysis of the hemicellulosefraction to constitutent monosaccharides. These sugars can be completelyliberated from the biomass with further treatment. In some embodiments,the further treatment is a hydrothermal treatment that includes washingthe exploded material with hot water, which removes contaminants such assalts. This step is not necessary for cellulosic ethanol fermentationsdue to the more dilute sugar concentrations used in such processes. Inother embodiments, the further treatment is additional acid treatment.In still other embodiments, the further treatment is enzymatichydrolysis of the exploded material. These treatments can also be usedin any combination. The type of treatment can affect the type of sugarsliberated (e.g., five carbon sugars versus six carbon sugars) and thestage at which they are liberated in the process. As a consequence,different streams of sugars, whether they are predominantly five-carbonor six-carbon, can be created. These enriched five-carbon or six-carbonstreams can thus be directed to specific microorganisms with differentcarbon utilization cabilities.

The methods of the present invention typically involve fermentation tohigher cell densities than what is achieved in ethanol fermentation.Because of the higher densities of the cultures for heterotrophiccellulosic oil production, the fixed carbon source (e.g., the cellulosicderived sugar stream(s)) is preferably in a concentrated form. Theglucose level of the depolymerized cellulosic material is preferably atleast 300 g/liter, at least 400 g/liter, at least 500 g/liter or atleast 600 g/liter prior to the cultivation step, which is optionally afed batch cultivation in which the material is fed to the cells overtime as the cells grow and accumulate lipid. Cellulosic sugar streamsare not used at or near this concentration range in the production ofcellulosic ethanol. Thus, in order to generate and sustain the very highcell densities during the production of lignocellulosic oil, the carbonfeedstock(s) must be delivered into the heterotrophic cultures in ahighly concentrated form. However, any component in the feedstream thatis not a substrate for, and is not metabolized by, the oleaginousmicroorganism will accumulate in the bioreactor, which can lead toproblems if the component is toxic or inhibitory to production of thedesired end product. While ligin and lignin-derived by-products,carbohydrate-derived byproducts such as furfurals and hydroxymethylfurfurals and salts derived from the generation of the cellulosicmaterials (both in the explosion process and the subsequentneutralization process), and even non-metabolized pentose/hexose sugarscan present problems in ethanolic fermentations, these effects areamplified significantly in a process in which their concentration in theinitial feedstock is high. To achieve sugar concentrations in the 300g/L range (or higher) for six-carbon sugars that may be used in largescale production of lignocellulosic oil described in the presentinvention, the concentration of these toxic materials can be 20 timeshigher than the concentrations typically present in ethanolicfermentations of cellulosic biomass.

The explosion process treatment of the cellulosic material utilizessignificant amounts of sulfuric acid, heat and pressure, therebyliberating by-products of carbohydrates, namely furfurals andhydroxymethyl furfurals. Furfurals and hydroxymethyl furfurals areproduced during hydrolysis of hemicellulose through dehydration ofxylose into furfural and water. In some embodiments of the presentinvention, these by-products (e.g., furfurals and hydroxymethylfurfurals) are removed from the saccharified lignocellulosic materialprior to introduction into the bioreactor. In certain embodiments of thepresent invention, the process for removal of the by-products ofcarbohydrates is hydrothermal treatment of the exploded cellulosicmaterials. In addition, the present invention provides methods in whichstrains capable of tolerating compounds such as furfurals orhydroxymethyl furfurals are used for lignocellulosic oil production. Inanother embodiment, the present invention also provides methods andmicroorganisms that are not only capable of tolerating furfurals in thefermentation media, but are actually able to metabolize theseby-products during the production of lignocellulosic oil.

The explosion process also generates significant levels of salts. Forexample, typical conditions for explosion can result in conductivites inexcess of 5 mS/cm when the exploded cellulosic biomass is resuspended ata ratio of 10:1 water:solids (dry weight). In certain embodiments of thepresent invention, the diluted exploded biomass is subjected toenzymatic saccharification, and the resulting supernatant isconcentrated up to 25 fold for use in the bioreactor. The salt level (asmeasured by conductivity) in the concentrated sugar stream(s) can beunacceptably high (up to 1.5 M Na⁺ equivalents). Additional salts aregenerated upon neutralization of the exploded materials for thesubsequent enzymatic saccharification process as well. The presentinvention provides methods for removing these salts so that theresulting concentrated cellulosic sugar stream(s) can be used inheterotrophic processes for producing lignocellulosic oil. In someembodiments, the method of removing these salts is deionization withresins, such as, but not limited to, DOWEX Marathon MR3. In certainembodiments, the deionization with resin step occurs before sugarconcentration or pH adjustment and hydrothermal treatment of biomassprior to saccharification, or any combination of the preceding; in otherembodiments, the step is conducted after one or more of these processes.In other embodiments, the explosion process itself is changed so as toavoid the generation of salts at unacceptably high levels. For example,a suitable alternative to sulfuric acid (or other acid) explosion of thecellulosic biomass is mechanical pulping to render the cellulosicbiomass receptive to enzymatic hydrolysis (saccharification). In stillother embodiments, native strains of microorganisms resistant to highlevels of salts or genetically engineered strains with resistance tohigh levels of salts are used.

A preferred embodiment for the process of preparing of explodedcellulosic biomass for use in heterotrophic lignocellulosic oilproduction using oleaginous microbes. A first step comprises adjustingthe pH of the resuspended exploded cellulosic biomass to the range of5.0-5.3 followed by washing the cellulosic biomass three times. Thiswashing step can be accomplished by a variety of means including the useof desalting and ion exchange resins, reverse omosis, hydrothermaltreatment (as described above), or just repeated re-suspension andcentrifugation in deionized water. This wash step results in acellulosic stream whose conductivity is between 100-300 μS/cm and theremoval of significant amounts of furfurals and hydroxymethyl furfurals.Decants from this wash step can be saved to concentrate five-carbonsugars liberated from the hemicellulose fraction. A second stepcomprises enzymatic saccharification of the washed cellulosic biomass.In a preferred embodiment, Accellerase (Genencor) is used. A third stepcomprises the recovery of sugars via centrifugation or decanting andrinsing of the saccharified biomass. The resulting biomass (solids) isan energy dense, lignin rich component that can be used as fuel or sentto waste. The recovered sugar stream in the centrifugation/decanting andrinse process is collected. A fourth step comprises microfiltration toremove contaminating solids with recovery of the permeate. A fifth stepcomprises a concentration step which can be accomplished using a vacuumevaporator. This step can optionally include the addition of antifoamagents such as P′2000 (Sigma/Fluka), which is sometimes necessary due tothe protein content of the resulting sugar feedstock.

In another embodiment of the methods of the invention, the carbon sourceis glycerol, including acidulated and non-acidulated glycerol byproductfrom biodiesel transesterification. In one embodiment, the carbon sourceincludes glycerol and at least one other carbon source. In some cases,all of the glycerol and the at least one other fixed carbon source areprovided to the microorganism at the beginning of the fermentation. Insome cases, the glycerol and the at least one other fixed carbon sourceare provided to the microorganism simultaneously at a predeterminedratio. In some cases, the glycerol and the at least one other fixedcarbon source are fed to the microbes at a predetermined rate over thecourse of fermentation.

Some microalgae undergo cell division faster in the presence of glycerolthan in the presence of glucose (see PCT Pub. No. 2008/151149). In theseinstances, two-stage growth processes in which cells are first fedglycerol to rapidly increase cell density, and are then fed glucose toaccumulate lipids can improve the efficiency with which lipids areproduced. The use of the glycerol byproduct of the transesterificationprocess provides significant economic advantages when put back into theproduction process. Other feeding methods are provided as well, such asmixtures of glycerol and glucose. Feeding such mixtures also capturesthe same economic benefits. In addition, the invention provides methodsof feeding alternative sugars to microalgae such as sucrose in variouscombinations with glycerol.

In another embodiment of the methods of the invention, the carbon sourceis invert sugar. Invert sugar is produced by splitting the sucrose intoits monosaccharide components, fructose and glucose. Production ofinvert sugar can be achieved through several methods that are known inthe art. One such method is heating an aqueous solution of sucrose.Often, catalysts are employed in order to accelerate the conversion ofsucrose into invert sugar. These catalysts can be biological, forexample enzymes such as invertases and sucrases can be added to thesucrose to accelerate the hydrolysis reaction to produce invert sugar.Acid is an example of non-biological catalyst, when paired with heat,can accelerate the hydrolysis reaction. Once the invert sugar is made,it is less prone to crystallization compared to sucrose and thus,provides advantages for storage and in fed batch fermentation, which inthe case of heterotrophic cultivation of microbes, including microalgae,there is a need for concentrated carbon source. In one embodiment, thecarbon source is invert sugar, preferably in a concentrated form,preferably at least 800 g/liter, at least 900 g/liter, at least 1000g/liter or at least 1100 g/liter prior to the cultivation step, which isoptionally a fed batch cultivation. The invert sugar, preferably in aconcentrated form, is fed to the cells over time as the cells grow andaccumulate lipid.

In another embodiment of the methods of the invention, the carbon sourceis sucrose, including a complex feedstock containing sucrose, such asthick cane juice from sugar cane processing. Because of the higherdensities of the cultures for heterotrophic oil production, the fixedcarbon source (e.g., sucrose, glucose, etc.) is preferably in aconcentrated form, preferably at least 500 g/liter, at least 600g/liter, at least 700 g/liter or at least 800 g/liter of the fixedcarbon source prior to the cultivation step, which is optionally a fedbatch cultivation in which the material is fed to the cells over time asthe cells grow and accumulate lipid. In the some cases, the carbonsource is sucrose in the form of thick cane juice, preferably in aconcentrated form, preferably at least 60% solids or about 770 g/litersugar, at least 70% solids or about 925 g/liter sugar, or at least 80%solids or about 1125 g/liter sugar prior to the cultivation step, whichis optionally a fed batch cultivation. The concentrated thick cane juiceis fed to the cells over time as the cells grow and accumulate lipid.

In one embodiment, the culture medium further includes at least onesucrose utilization enzyme. In some cases, the culture medium includes asucrose invertase. In one embodiment, the sucrose invertase enzyme is asecrectable sucrose invertase enzyme encoded by an exogenous sucroseinvertase gene expressed by the population of microorganisms. Thus, insome cases, as described in more detail in Section IV, below, themicroalgae has been genetically engineered to express a sucroseutilization enzyme, such as a sucrose transporter, a sucrose invertase,a hexokinase, a glucokinase, or a fructokinase.

Complex feedstocks containing sucrose include waste molasses from sugarcane processing; the use of this low-value waste product of sugar caneprocessing can provide significant cost savings in the production ofhydrocarbons and other oils. Another complex feedstock containingsucrose that is useful in the methods of the invention is sorghum,including sorghum syrup and pure sorghum. Sorghum syrup is produced fromthe juice of sweet sorghum cane. Its sugar profile consists of mainlyglucose (dextrose), fructose and sucrose.

4. Oil Production

For the production of oil in accordance with the methods of theinvention, it is preferable to culture cells in the dark, as is thecase, for example, when using extremely large (40,000 liter and higher)fermentors that do not allow light to strike the culture. Protothecaspecies are grown and propagated for the production of oil in a mediumcontaining a fixed carbon source and in the absence of light; suchgrowth is known as heterotrophic growth.

As an example, an inoculum of lipid-producing oleaginous microbialcells, preferably microalgal cells are introduced into the medium; thereis a lag period (lag phase) before the cells begin to propagate.Following the lag period, the propagation rate increases steadily andenters the log, or exponential, phase. The exponential phase is in turnfollowed by a slowing of propagation due to decreases in nutrients suchas nitrogen, increases in toxic substances, and quorum sensingmechanisms. After this slowing, propagation stops, and the cells enter astationary phase or steady growth state, depending on the particularenvironment provided to the cells. For obtaining lipid rich biomass, theculture is typically harvested well after then end of the exponentialphase, which may be terminated early by allowing nitrogen or another keynutrient (other than carbon) to become depleted, forcing the cells toconvert the carbon sources, present in excess, to lipid. Culturecondition parameters can be manipulated to optimize total oilproduction, the combination of lipid species produced, and/or productionof a specific oil.

As discussed above, a bioreactor or fermentor is used to allow cells toundergo the various phases of their growth cycle. As an example, aninoculum of lipid-producing cells can be introduced into a mediumfollowed by a lag period (lag phase) before the cells begin growth.Following the lag period, the growth rate increases steadily and entersthe log, or exponential, phase. The exponential phase is in turnfollowed by a slowing of growth due to decreases in nutrients and/orincreases in toxic substances. After this slowing, growth stops, and thecells enter a stationary phase or steady state, depending on theparticular environment provided to the cells. Lipid production by cellsdisclosed herein can occur during the log phase or thereafter, includingthe stationary phase wherein nutrients are supplied, or still available,to allow the continuation of lipid production in the absence of celldivision.

Preferably, microorganisms grown using conditions described herein andknown in the art comprise at least about 20% by weight of lipid,preferably at least about 40% by weight, more preferably at least about50% by weight, and most preferably at least about 60% by weight. Processconditions can be adjusted to increase the yield of lipids suitable fora particular use and/or to reduce production cost. For example, incertain embodiments, a microalgae is cultured in the presence of alimiting concentration of one or more nutrients, such as, for example,nitrogen, phosphorous, or sulfur, while providing an excess of fixedcarbon energy such as glucose. Nitrogen limitation tends to increasemicrobial lipid yield over microbial lipid yield in a culture in whichnitrogen is provided in excess. In particular embodiments, the increasein lipid yield is at least about: 10%, 50%, 100%, 200%, or 500%. Themicrobe can be cultured in the presence of a limiting amount of anutrient for a portion of the total culture period or for the entireperiod. In particular embodiments, the nutrient concentration is cycledbetween a limiting concentration and a non-limiting concentration atleast twice during the total culture period. Lipid content of cells canbe increased by continuing the culture for increased periods of timewhile providing an excess of carbon, but limiting or no nitrogen.

In another embodiment, lipid yield is increased by culturing alipid-producing microbe (e.g., microalgae) in the presence of one ormore cofactor(s) for a lipid pathway enzyme (e.g., a fatty acidsynthetic enzyme). Generally, the concentration of the cofactor(s) issufficient to increase microbial lipid (e.g., fatty acid) yield overmicrobial lipid yield in the absence of the cofactor(s). In a particularembodiment, the cofactor(s) are provided to the culture by including inthe culture a microbe (e.g., microalgae) containing an exogenous geneencoding the cofactor(s). Alternatively, cofactor(s) may be provided toa culture by including a microbe (e.g., microalgae) containing anexogenous gene that encodes a protein that participates in the synthesisof the cofactor. In certain embodiments, suitable cofactors include anyvitamin required by a lipid pathway enzyme, such as, for example:biotin, pantothenate. Genes encoding cofactors suitable for use in theinvention or that participate in the synthesis of such cofactors arewell known and can be introduced into microbes (e.g., microalgae), usingcontructs and techniques such as those described above.

The specific examples of bioreactors, culture conditions, andheterotrophic growth and propagation methods described herein can becombined in any suitable manner to improve efficiencies of microbialgrowth and lipid and/or protein production.

Microalgal biomass with a high percentage of oil/lipid accumulation bydry weight has been generated using different methods of culture, whichare known in the art (see PCT Pub. No. 2008/151149). Microalgal biomassgenerated by the culture methods described herein and useful inaccordance with the present invention comprises at least 10% microalgaloil by dry weight. In some embodiments, the microalgal biomass comprisesat least 25%, at least 50%, at least 55%, or at least 60% microalgal oilby dry weight. In some embodiments, the microalgal biomass contains from10-90% microalgal oil, from 25-75% microalgal oil, from 40-75%microalgal oil, or from 50-70% microalgal oil by dry weight.

The microalgal oil of the biomass described herein, or extracted fromthe biomass for use in the methods and compositions of the presentinvention can comprise glycerolipids with one or more distinct fattyacid ester side chains. Glycerolipids are comprised of a glycerolmolecule esterified to one, two or three fatty acid molecules, which canbe of varying lengths and have varying degrees of saturation. The lengthand saturation characteristics of the fatty acid molecules (and themicroalgal oils) can be manipulated to modify the properties orproportions of the fatty acid molecules in the microalgal oils of thepresent invention via culture conditions or via lipid pathwayengineering, as described in more detail in Section IV, below. Thus,specific blends of algal oil can be prepared either within a singlespecies of algae by mixing together the biomass or algal oil from two ormore species of microalgae, or by blending algal oil of the inventionwith oils from other sources such as soy, rapeseed, canola, palm, palmkernel, coconut, corn, waste vegetable, Chinese tallow, olive,sunflower, cottonseed, chicken fat, beef tallow, porcine tallow,microalgae, macroalgae, microbes, Cuphea, flax, peanut, choice whitegrease, lard, Camelina sativa, mustard seed, cashew nut, oats, lupine,kenaf, calendula, help, coffee, linseed (flax), hazelnut, euphorbia,pumpkin seed, coriander, camellia, sesame, safflower, rice, tung tree,cocoa, copra, pium poppy, castor beans, pecan, jojoba, macadamia, Brazilnuts, avocado, petroleum, or a distillate fraction of any of thepreceding oils.

The oil composition, i.e., the properties and proportions of the fattyacid consitutents of the glycerolipids, can also be manipulated bycombining biomass or oil from at least two distinct species ofmicroalgae. In some embodiments, at least two of the distinct species ofmicroalgae have different glycerolipid profiles. The distinct species ofmicroalgae can be cultured together or separately as described herein,preferably under heterotrophic conditions, to generate the respectiveoils. Different species of microalgae can contain different percentagesof distinct fatty acid consituents in the cell's glycerolipids.

Generally, Prototheca strains have very little or no fatty acids withthe chain length C8-C14. For example, Prototheca moriformis (UTEX 1435),Prototheca krugani (UTEX 329), Prototheca stagnora (UTEX 1442) andPrototheca zopfii (UTEX 1438) contains no (or undectable amounts) C8fatty acids, between 0-0.01% C10 fatty acids, between 0.03-2.1% C12fatty acids and between 1.0-1.7% C14 fatty acids.

In some cases, the Prototheca strains containing a transgene encoding afatty acyl-ACP thioesterase that has activity towards fatty acyl-ACPsubstrate of chain lengths C8 or C8-10 has at least 1%, at least 1.5%,at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, atleast 12%, or at least 15% or more, fatty acids of chain length C8. Inother instances, the Prototheca strains containing a transgene encodinga fatty acyl ACP thioesterase that has activity towards fatty acyl-ACPsubstrate of chain lengths C10 has at least at least 1%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 24%, or at least 25% ormore, fatty acids of chain length C10. In other instances, thePrototheca strains containing a transgene encoding a fatty acyl-ACPthioesterase that has activity towards fatty acyl-ACP substrate of chainlength C12 has at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 34%, at least 35% or atleast 40% or more, fatty acids of the chain length C12. In other cases,the Prototheca strains containing a transgene encoding a fatty acyl-ACPthioesterase that has activity towards fatty acyl-ACP substrate of chainlength C14 has at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 7%, at least 10%, at least 15%, at least30%, at least 43%, or at least 45% or more, fatty acids of the chainlength C14.

In non-limiting examples, the Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has activity towards fattyacyl-ACP substrate of chain length C8 has between 1%-25%, or between1%-15%, preferably 1.8-12.29%, fatty acids of chain length C8. In othernon-limiting examples, Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has activity towards fattyacyl-ACP substrate of chain length C10 has between 1%-50%, or between1%-25%, preferably 1.91-23.97% fatty acids of chain length C10. In othernon-limiting examples, Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has activity towards fattyacyl-ACP substrate of chain length C12 has between 5%-50%, or between10%-40, preferably 13.55-34.01%, fatty acids of the chain length C12. Inother non-limiting examples, Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has activity towards fattyacyl-ACP substrate of chain length C14 has between 1%-60%, or between2%-45%, preferably 2.59-43.27%, fatty acids of the chain length C14. Inother non-limiting examples, Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has broad specificitytowards fatty acyl-ACP substrates of varying carbon chain length has upto 30%, up to 35%, or preferably up to 39.45% fatty acids of the chainlength C16. In some cases, the Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has activity towards fattyacyl-ACP substrate of chain lengths between C8 and C14 have between1%-75%, or between 2%-60%, preferably 2.69-57.98%, medium chain (C8-C14)fatty acids. In some cases, the Prototheca strains containing atransgene encoding a fatty acyl-ACP thioesterase that has activitytowards fatty acyl-ACP substrates of chain lengths between C12 and C14have at least 30%, at least 40%, or at least 49% C12-C14 fatty acids. Insome instances, keeping the transgenic Prototheca strains under constantand high selective pressure to retain exogenous genes is advantageousdue to the increase in the desired fatty acid of a specific chainlength. High levels of exogenous gene retention can also be achieved byinserting exogenous genes into the nuclear chromosomes of the cellsusing homologous recombination vectors and methods disclosed herein.Recombinant cells containing exogenous genes integrated into nuclearchromosomes are an object of the invention.

Microalgal oil can also include other constituents produced by themicroalgae, or incorporated into the microalgal oil from the culturemedium. These other constituents can be present in varying amountdepending on the culture conditions used to culture the microalgae, thespecies of microalgae, the extraction method used to recover microalgaloil from the biomass and other factors that may affect microalgal oilcomposition. Non-limiting examples of such constituents includecarotenoids, present from 0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably0.05 to 0.244 micrograms/gram, of oil; chlorophyll A present from0.01-0.5 mcg/g, 0.025-0.3 mcg/g, preferably 0.045 to 0.268micrograms/gram, of oil; total chlorophyll of less than 0.1 mcg/g, lessthan 0.05 mcg/g, preferably less than 0.025 micrograms/gram, of oil;gamma tocopherol present from 1-300 mcg/g, 35-175 mcg/g, preferably38.3-164 micrograms/gram, of oil; total tocopherols present from 10-500mcg/g, 50-300 mcg/g, preferably 60.8 to 261.7 microgram/gram, of oil;less than 1%, less than 0.5%, preferably less than 0.25% brassicasterol,campesterol, stigmasterol, or betasitosterol; total tocotrienols lessthan 400 mcg/g, preferably less than 300 micrograms/gram, of oil; ortotal tocotrienols present from 100-500 mcg/g, 225-350 mcg/g, preferably249.6 to 325.3 micrograms/gram, of oil.

The other constituents can include, without limitation, phospholipids,tocopherols, tocotrienols, carotenoids (e.g., alpha-carotene,beta-carotene, lycopene, etc.), xanthophylls (e.g., lutein, zeaxanthin,alpha-cryptoxanthin and beta-crytoxanthin), and various organic orinorganic compounds. In some cases, the oil extracted from Protothecaspecies comprises between 0.001-0.01 mcg/g, 0.0025-0.05 mcg/g,preferably 0.003 to 0.039 microgram lutein/gram, of oil, less than 0.01mcg/g, less than 0.005 mcg/g, preferably less than 0.003 microgramslycopene/gram, of oil; and less than 0.01 mcg/g, less than 0.005 mcg/g,preferably less than 0.003 microgram beta carotene/gram, of oil.

In some embodiments, the present invention provides an oleaginousmicrobial cell comprising a triglyceride oil, wherein the fatty acidprofile of the triglyceride oil is selected from the group consistingof: at least about 1%, at least about 2%, at least about 5%, at leastabout 7%, at least about 10%, or at least about 15%, C8:0; at leastabout 1%, at least about 5%, at least about 15%, at least about 20%, atleast about 25%, or at least about 30%, C10:0; at least about 1%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, or at least about 80%, C12:0; at least about 2%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, or at least about 50%, C14:0; atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, or at least about 90%, C16:0; at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, or at least about 50%, C18:0; at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, or at least about 90%, C18:1; less than about7%, less than about 5%, less than about 3%, less than about 1%, or about0%, C18:2; and at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, or at least about 90%, saturated fatty acids.

In some embodiments, the oleaginous microbial cell comprisestriglyceride oil comprising a fatty acid profile selected from the groupconsisting of: total combined amounts of C8:0 and C10:0 of at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or about 100%; total combined amounts ofC10:0, C12:0, and C14:0 of at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or about 100%;total combined amounts of C16:0, C18:0 and C18:1 of at least about 60%,at least about 70%, at least about 80%, at least about 90%, or about100%; total combined amounts of C18:0, C18:1 and C18:2 of at least about60%, at least about 70%, at least about 80%, at least about 90%, orabout 100%; total combined amounts of C14:0, C16:0, C18:0 and C18:1 ofat least about 60%, at least about 70s %, at least about 80%, at leastabout 90%, or about 100%; and total combined amounts of C18:1 and C18:2of less than about 30%, less than about 25%, less than about 20%, lessthan about 15%, less than about 10%, less than about 5%, or about 0%,

In some embodiments, the oleaginous microbial cell comprisestriglyceride oil having a fatty acid profile comprising a ratio of fattyacids selected from the group consisting of: a C8:0 to C10:0 ratio of atleast about 5 to 1, at least 6 to 1, at least 7 to 1, at least 8 to 1,at least 9 to 1, or at least 10 to 1; a C10:0 to C12:0 ratio of at leastabout 6 to 1, at least 7 to 1, at least 8 to 1, at least 9 to 1, or atleast 10 to 1; a C12:0 to C14:0 ratio of at least about 5 to 1, at least6 to 1, at least 7 to 1, at least 8 to 1, at least 9 to 1, or at least10 to 1; a C14:0 to C12:0 ratio of at least 7 to 1, at least 8 to 1, atleast 9 to 1, or at least 10 to 1; and a C14:0 to C16:0 ratio of atleast 1 to 2, at least 1 to 3, at least 1 to 4, at least 1 to 5, atleast 1 to 6, at least 1 to 7, at least 1 to 8, at least 1 to 9, or atleast 1 to 10.

In some embodiments, the present invention provides an oleaginousmicrobial triglyceride oil composition, wherein the fatty acid profileof the triglyceride oil is selected from the group consisting of: atleast about 1%, at least about 2%, at least about 5%, at least about 7%,at least about 10%, or at least about 15%, C8:0; at least about 1%, atleast about 5%, at least about 15%, at least about 20%, at least about25%, or at least about 30% C10:0; at least about 1%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,or at least about 80%, C12:0; at least about 2%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, or at least about 50%, C14:0; at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, or at least about 90%, C16:0; at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, or at least about 50%, C18:0; at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, or at least about 90%, C18:1; less than about 7%, lessthan about 5%, less than about 3%, less than about 1%, or about 0%,C18:2; and at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, or at least about 90%, saturated fatty acids.

In some embodiments, the oleaginous microbial triglyceride oilcomposition comprises triglyceride oil comprising a fatty acid profilein which: the total combined amount of C10:0, C12:0 and C14:0 is atleast about 50%, at least bout 60%, at least about 70%, at least about80%, at least about 90%, or about 100%; the total combined amount ofC16:0, C18:0 and C18:1 is at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or about 100%; the total combinedamount of C18:0, C18:1 and C18:2 is at least about 60%, at least about70%, at least about 80%, at least about 90%, or about 100%; the totalcombined amount of C14:0, C16:0, C18:0 and C18:1 is at least about 60%,at least about 70%, at least about 80%, at least about 90%, or about100%; the total combined amounts of C8:0 and C10:0 is less than about50%, less than about 45%, less than about 40%, less than about 35%, lessthan about 30%, less than about 25%, less than about 20%, less thanabout 15%, less than about 10%, less than about 5%, or about 0%.

In some embodiments, the oleaginous microbial triglyceride oilcomposition comprises triglyceride oil having a fatty acid profilecomprising a ratio of fatty acids selected from the group consisting of:a C8:0 to C10:0 ratio of at least about 5 to 1, at least about 6 to 1,at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, orat least about 10 to 1; a C10:0 to C12:0 ratio of at least about 6 to 1,at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, orat least about 10 to 1; a C12:0 to C14:0 ratio of at least about 5 to 1,at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, atleast about 9 to 1, or at least about 10 to 1; a C14:0 to C12:0 ratio ofat least about 7 to 1, at least about 8 to 1, at least about 9 to 1, orat least about 10 to 1; a C14:0 to C16:0 ratio of at least about 1 to 2,at least about 1 to 3, at least about 1 to 4, at least about 1 to 5, atleast about 1 to 6, at least about 1 to 7, at least about 1 to 8, atleast about 1 to 9, or at least about 1 to 10.

In some embodiments, the present invention provides a method ofproducing an oleaginous microbial triglyceride oil composition having afatty acid profile selected from the group consisting of: at least about1%, at least about 2%, at least about 5%, at least about 7%, at leastabout 10%, or at least about 15%, C8:0; at least about 1%, at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, or at least about 30%, C10:0; at least about 1%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, or at least about 80%, C12:0; at least about 2%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, or at least about 50%, C14:0; atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, or at least about 90%, C16:0; at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, or at least about 50% C18:0; at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, or at least about 90%, C18:1; less than about7%, less than about 5%, less than about 3%, less than about 1%, or about0%, C18:2; and at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, or at least about 90%, saturated fatty acids,wherein the method comprises the steps of: (a) cultivating a populationof oleaginous microbial cells in a culture medium until at least 10% ofthe dry cell weight of the oleaginous microbial cells is triglycerideoil; and (b) isolating the triglyceride oil composition from theoleaginous microbial cells.

In some embodiments, the method of producing oleaginous microbialtriglyceride oil compositions yields triglyceride oils comprising afatty acid profile in which: the total combined amount of C10:0, C12:0and C14:0 is at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, or about 100%; the totalcombined amount of C16:0, C18:0 and C18:1 is at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or about 100%;the total combined amount of C18:0, C18:1 and C18:2 is at least about60%, at least about 70%, at least about 80%, at least about 90%, orabout 100%; the total combined amount of C14:0, C16:0, C18:0 and C18:1is at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, or about 100%; the total combined amount of C8:0 and C10:0 isless than about 50%, less than about 45%, less than about 40%, less thanabout 35%, less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, orabout 0%.

In some embodiments, the method of producing oleaginous microbialtriglyceride oil compositions yields triglyceride oils having a fattyacid profile comprising a ratio of triglyceride oils selected from thegroup consisting of: a C8:0 to C10:0 ratio of at least about 5 to 1, atleast about 6 to 1, at least about 7 to 1, at least about 8 to 1, atleast about 9 to 1, or at least about 10 to 1; a C10:0 to C12:0 ratio ofat least about 6 to 1, at least about 7 to 1, at least about 8 to 1, atleast about 9 to 1, or at least about 10 to 1; a C12:0 to C14:0 ratio ofat least about 5 to 1, at least about 6 to 1, at least about 7 to 1, atleast about 8 to 1, at least about 9 to 1, or at least about 10 to 1; aC14:0 to C12:0 ratio of at least about 7 to 1, at least about 8 to 1, atleast about 9 to 1, or at least about 10 to 1; and a C14:0 to C16:0ratio of at least about 1 to 2, at least about 1 to 3, at least about 1to 4, at least about 1 to 5, at least about 1 to 6, at least about 1 to7, at least about 1 to 8, at least about 1 to 9, or at least about 1 to10.

III. Genetic Engineering Methods and Materials

The present invention provides methods and materials for genenticallymodifying microorganisms, including Prototheca cells and recombinanthost cells, useful in the methods of the present invention, includingbut not limited to recombinant Prototheca moriformis, Prototheca zopfii,Prototheca krugani, and Prototheca stagnora host cells. The descriptionof these methods and materials is divided into subsections for theconvenience of the reader. In subsection 1, transformation methods aredescribed. In subsection 2, genetic engineering methods using homologousrecombination are described. In subsection 3, expression vectors andcomponents are described.

In certain embodiments of the present invention it is desirable togenentically modify a microorganism to enhance lipid production, modifythe properties or proportions of components generated by themicroorganism, or to improve or provide de novo growth characteristicson a variety of feedstock materials. Chlorella, particularly Chlorellaprotothecoides, Chlorella minutissima, Chlorella sorokiniana, Chlorellaellipsoidea, Chlorella sp., and Chlorella emersonii are preferredmicroorganisms for use in the genetic engineering methods describedherein, although other Chlorella species as well as other varieties ofmicroorganisms can be used.

Promoters, cDNAs, and 3′UTRs, as well as other elements of the vectors,can be generated through cloning techniques using fragments isolatedfrom native sources (see for example Molecular Cloning: A LaboratoryManual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press; andU.S. Pat. No. 4,683,202). Alternatively, elements can be generatedsynthetically using known methods (see for example Gene. 1995 Oct. 16;164(1):49-53).

1. Engineering Methods—Transformation

Cells can be transformed by any suitable technique including, e.g.,biolistics, electroporation (see Maruyama et al. (2004), BiotechnologyTechniques 8:821-826), glass bead transformation and silicon carbidewhisker transformation. Another method that can be used involves formingprotoplasts and using CaCl₂ and polyethylene glycol (PEG) to introducerecombinant DNA into microalgal cells (see Kim et al. (2002), Mar.Biotechnol. 4:63-73, which reports the use of this method for thetransformation of Chorella ellipsoidea). Co-transformation of microalgaecan be used to introduce two distinct vector molecules into a cellsimultaneously (see for example Protist 2004 December; 155(4):381-93).

Biolistic methods (see, for example, Sanford, Trends In Biotech. (1988)6:299 302, U.S. Pat. No. 4,945,050; electroporation (Fromm et al., Proc.Nat'l. Acad. Sci. (USA) (1985) 82:5824 5828); use of a laser beam,microinjection or any other method capable of introducing DNA into amicroalgae can also be used for transformation of a Prototheca cell.

Any convenient technique for introducing a transgene into amicroorganism, such as Chorella, can be employed in the presentinvention. Dawson et al. (1997) (supra) described the use ofmicro-projectile bombardment to introduce the nitrate reductase (NR)gene from Chlorella vulgaris into NR-deficient Chlorella sorokinianamutants, resulting in stable transformants. Briefly, 0.4 micron tungstenbeads were coated with plasmid; 3×10⁷ C. sorokiniana cells were spreadin the center third of a non-selective agar plate and bombarded with thePDS-1000/He Biolistic Particle Delivery® system (Bio-Rad).

A preferred method for introducing a transgene into a microorganism,such as Chlorella, is the method described by Kim et al. (2002), Mar.Biotechnol. 4:63-73. Kim reports the transformation of Chorellaellipsoidea protoplasts using CaCl₂ and polyethylene glycol (PEG). Inparticular, protoplasts were prepared by growing C. ellipsoidea cells toa density of 1−2×10⁸/Ml. Cells were recovered and washed bycentrifugation for 5 minutes at 1600 g and resuspended in 5 Ml ofphosphate buffer (Ph 6.0) containing 0.6 M sorbitol, 0.6 M mannitol, 4%(weight/volume) cellulose (Calbiochem), 2% (weight/volume) macerase(Calbiochem), and 50 units pectinase (Sigma). The cell suspension wasincubated at 25° C. for 16 hours in the dark with gentle shaking. Theresultant protoplasts were recovered by centrifugation at 400 g for 5minutes. The pellet was gently resuspended in 5 Ml of f/2 mediumcontaining 0.6 M sorbitol and 0.6 M mannitol and centrifuged at 400 gfor 5 minutes. This pellet was resuspended in 1 Ml of 0.6 Msorbitol/mannitol solution containing 50 mMCaCl₂. Then, 5 mg oftransgene DNA was added, along with 25 μg calf thymus DNA (Sigma), to10⁷-10⁸ protoplasts in 0.4 Ml. After 15 minutes at room temperature, 200μL it of PNC (40% polyethylene glycol 4000, 0.8 M NaCl, 50 Mm CaCl₂) wasadded and mixed gently for 30 minutes at room temperature. After this,0.6 Ml of f/2 medium supplemented with 0.6 M sorbitol/mannitol solution,1% yeast extract and 1% glucose was added, and the transformed cellswere incubated at 25° C. for 12 hours in the dark for cell wallregeneration. A similar method was used by Huang et al. (2007) (supra)to introduce a transgene encoding mercuric reductase into Chlorella sp.DT.

Electorporation has also been employed to transform microorganisms, suchas Chorella. As reported by Maruyama et al. (2004), BiotechnologyTechniques 8:821-826 (incorporated by reference herein in its entirety),this technique was used to introduce a transgene into protoplasts ofChlorella saccharophila c-211-1a prepared from the cells in thestationary phase. Transient expression of the introduced plasmid wasobserved under a field strength of between 600 and 900 V/cm, and a pulseduration of around 400 ms, where high membrane permeability to 70-kDaFITC-dextran was ascertained.

Examples of expression of transgenes in microorganisms, such asChlorella, can be found in the literature (see for example CurrentMicrobiology Vol. 35 (1997), pp. 356-362; Sheng Wu Gong Cheng Xue Bao.2000 July; 16(4):443-6; Current Microbiology Vol. 38 (1999), pp.335-341; Appl Microbiol Biotechnol (2006) 72: 197-205; MarineBiotechnology 4, 63-73, 2002; Current Genetics 39:5, 365-370 (2001);Plant Cell Reports 18:9, 778-780, (1999); Biologia Plantarium 42(2):209-216, (1999); Plant Pathol. J 21(1): 13-20, (2005)). Also seeExamples herein.

Examples of expression of transgenes in oleaginous yeast (e.g., Yarrowialipolytica) can be found in the literature (see, for example, Bordes etal., J Microbiol Methods, Jun. 27 (2007)). Examples of expression oftransgenes in fungi (e.g., Mortierella alpine, Mucor circinelloides, andAspergillus ochraceus) can also be found in the literature (see, forexample, Microbiology, July; 153(Pt. 7):2013-25 (2007); Mol GenetGenomics, June; 271(5):595-602 (2004); Curr Genet, March; 21(3):215-23(1992); Current Microbiology, 30(2):83-86 (1995); Sakuradani, NISRResearch Grant, “Studies of Metabolic Engineering of UsefulLipid-producing Microorganisms” (2004); and PCT/JP2004/012021). Examplesof expression of exogenous genes in bacteria such as E. coli are wellknown; see for example Molecular Cloning: A Laboratory Manual, Sambrooket al. (3d edition, 2001, Cold Spring Harbor Press.

Vectors for transformation of microorganisms in accordance with thepresent invention can be prepared by known techniques familiar to thoseskilled in the art. The nucleotide sequence of the construct used fortransformation of multiple Chlorella species corresponds to SEQ ID NO:8. In one embodiment, an exemplary vector design for expression of alipase gene in a microorganism such as a microalgae contains a geneencoding a lipase in operable linkage with a promoter active inmicroalgae. Alternatively, if the vector does not contain a promoter inoperable linkage with the gene of interest, the gene can be transformedinto the cells such that it becomes operably linked to an endogenouspromoter at the point of vector integration. The promoterless method oftransformation has been proven to work in microalgae (see for examplePlant Journal 14:4, (1998), pp. 441-447). The vector can also contain asecond gene that encodes a protein that, e.g., imparts resistance to anantibiotic or herbicide, i.e., a selectable marker. Optionally, one orboth gene(s) is/are followed by a 3′ untranslated sequence containing apolyadenylation signal. Expression cassettes encoding the two genes canbe physically linked in the vector or on separate vectors.Co-transformation of microalgae can also be used, in which distinctvector molecules are simultaneously used to transform cells (see forexample Protist 2004 December; 155(4):381-93). The transformed cells canbe optionally selected based upon the ability to grow in the presence ofthe antibiotic or other selectable marker under conditions in whichcells lacking the resistance cassette would not grow.

2. Engineering Methods—Homologous Recombination

Homologous recombination is the ability of complementary DNA sequencesto align and exchange regions of homology. Transgenic DNA (“donor”)containing sequences homologous to the genomic sequences being targeted(“template”) is introduced into the organism and then undergoesrecombination into the genome at the site of the corresponding genomichomologous sequences. The mechanistic steps of this process, in mostcasees, include: (1) pairing of homologous DNA segments; (2)introduction of double-stranded breaks into the donor DNA molecule; (3)invasion of the template DNA molecule by the free donor DNA endsfollowed by DNA synthesis; and (4) resolution of double-strand breakrepair events that result in final recombination products.

The ability to carry out homologous recombination in a host organism hasmany practical implications for what can be carried out at the moleculargenetic level and is useful in the generation of an oleaginous microbethat can produced tailored oils. By its very nature homologousrecombination is a precise gene targeting event, hence, most transgeniclines generated with the same targeting sequence will be essentiallyidentical in terms of phenotype, necessitating the screening of farfewer transformation events. Homologous recombination also targets geneinsertion events into the host chromosome, resulting in excellentgenetic stability, even in the absence of genetic selection. Becausedifferent chromosomal loci will likey impact gene expression, even fromheterologous promoters/UTRs, homologous recombination can be a method ofquerying loci in an unfamiliar genome environment and to assess theimpact of these environments on gene expression.

Particularly useful genetic engineering applications using homologousrecombination is to co-opt specific host regulatory elements such aspromoters/UTRs to drive heterologous gene expression in a highlyspecific fashion. For example, ablation or knockout of desaturasegenes/gene families with a heterologous gene encoding a selective markermight be expected to increase overall percentage of saturated fattyacids produced in the host cell. Example 11 describes the homologousrecombination targeting constructs and a working example of suchdesaturase gene ablations or knockouts generated in Protothecamoriformis.

Because homologous recombination is a precise gene targeting event, itcan be used to precisely modify any nucleotide(s) within a gene orregion of interest, so long as sufficient flanking regions have beenidentified. Therefore, homologous recombination can be used as a meansto modify regulatory sequences impacting gene expression of RNA and/orproteins. It can also be used to modify protein coding regions in aneffort to modify enzyme activities such as substrate specificity,affinities and Km, and thus affecting the desired change in metabolismof the host cell. Homologous recombination provides a powerful means tomanipulate the gost genome resulting in gene targeting, gene conversion,gene deletion, gene duplication, gene inversion and exchanging geneexpression regulatory elements such as promoters, enhancers and 3′UTRs.

Homologous recombination can be achieve by using targeting constructscontaining pieces of endogenous sequences to “target” the gene or regionof interest within the endogenous host cell genome. Such targetingsequences can either be located 5′ of the gene or region of interest, 3′of the gene/region of interest or even flank the gene/region ofinterest. Such targeting constructs can be transformed into the hostcell either as a supercoiled plasmid DNA with additional vectorbackbone, a PCR product with no vector backbone, or as a linearizedmolecule. In some cases, it may be advantageous to first expose thehomologous sequences within the transgenic DNA (donor DNA) with arestriction enzyme. This step can increase the recombination efficiencyand decrease the occurance of undesired events. Other methods ofincreasing recombination efficiency include using PCR to generatetransforming transgenic DNA containing linear ends homologous to thegenomic sequences being targeted.

For purposes of non-limiting illustration, regions of donor DNAsequences that are useful for homologous recombination include the KE858region of DNA in Prototheca moriformis. KE858 is a 1.3 kb, genomicfragment that encompasses part of the coding region for a protein thatshares homology with the transfer RNA (tRNA) family of proteins.Southern blots have shown that the KE858 sequence is present in a singlecopy in the Prototheca moriformis (UTEX 1435) genome. This region andExamples of using this region for homologous recombination targeting hasbeen described in PCT Application No. PCT/US2009/66142. Another regionof donor DNA that is useful is portions of the 6S rRNA genomic sequence.The use of this sequence in homologous recombination in Protothecamorifomis are described below in the Examples.

3. Vectors and Vector Components

Vectors for transformation of microorganisms in accordance with thepresent invention can be prepared by known techniques familiar to thoseskilled in the art in view of the disclosure herein. A vector typicallycontains one or more genes, in which each gene codes for the expressionof a desired product (the gene product) and is operably linked to one ormore control sequences that regulate gene expression or target the geneproduct to a particular location in the recombinant cell. To aid thereader, this subsection is divided into subsections. Subsection Adescribes control sequences typically contained on vectors as well asnovel control sequences provided by the present invention. Subsection Bdescribes genes typically contained in vectors as well as novel codonoptimization methods and genes prepared using them provided by theinvention.

A. Control Sequences

Control sequences are nucleic acids that regulate the expression of acoding sequence or direct a gene product to a particular location in oroutside a cell. Control sequences that regulate expression include, forexample, promoters that regulate transcription of a coding sequence andterminators that terminate transcription of a coding sequence. Anothercontrol sequence is a 3′ untranslated sequence located at the end of acoding sequence that encodes a polyadenylation signal. Control sequencesthat direct gene products to particular locations include those thatencode signal peptides, which direct the protein to which they areattached to a particular location in or outside the cell.

Thus, an exemplary vector design for expression of an exogenous gene ina microalgae contains a coding sequence for a desired gene product (forexample, a selectable marker, a lipid pathway modification enzyme, or asucrose utilization enzyme) in operable linkage with a promoter activein microalgae. Alternatively, if the vector does not contain a promoterin operable linkage with the coding sequence of interest, the codingsequence can be transformed into the cells such that it becomes operablylinked to an endogenous promoter at the point of vector integration. Thepromoterless method of transformation has been proven to work inmicroalgae (see for example Plant Journal 14:4, (1998), pp. 441-447).

Many promoters are active in microalgae, including promoters that areendogenous to the algae being transformed, as well as promoters that arenot endogenous to the algae being transformed (i.e., promoters fromother algae, promoters from higher plants, and promoters from plantviruses or algae viruses). Illustrative exogenous and/or endogenouspromoters that are active in microalgae (as well as antibioticresistance genes functional in microalgae) are described in PCT Pub. No.2008/151149 and references cited therein).

The promoter used to express an exogenous gene can be the promoternaturally linked to that gene or can be a heterologous gene. Somepromoters are active in more than one species of microalgae. Otherpromoters are species-specific. Illustrative promoters include promoterssuch as β-tubulin from Chlamydomonas reinhardtii, used in the Examplesbelow, and viral promoters, such as cauliflower mosaic virus (CMV) andchlorella virus, which have been shown to be active in multiple speciesof microalgae (see for example Plant Cell Rep. 2005 March;23(10-11):727-35; J Microbiol. 2005 August; 43(4):361-5; Mar Biotechnol(NY). 2002 January; 4(1):63-73). Another promoter that is suitable foruse for expression of exogenous genes in Prototheca is the Chlorellasorokiniana glutamate dehydrogenase promoter/5′UTR. Optionally, at least10, 20, 30, 40, 50, or 60 nucleotides or more of these sequencescontaining a promoter are used. Illustrative promoters useful forexpression of exogenous genes in Prototheca are listed in the sequencelisting of this application, such as the promoter of the Chlorella HUP1gene (SEQ ID NO:1) and the Chlorella ellipsoidea nitrate reductasepromoter (SEQ ID NO:2). Chlorella virus promoters can also be used toexpress genes in Prototheca, such as SEQ ID NOs: 1-7 of U.S. Pat. No.6,395,965. Additional promoters active in Prototheca can be found, forexample, in Biochem Biophys Res Commun 1994 Oct. 14; 204(1):187-94;Plant Mol. Biol. 1994 October; 26(1):85-93; Virology. 2004 Aug. 15;326(1):150-9; and Virology. 2004 Jan. 5; 318(1):214-23. Other usefulpromoters are described in detail in the Examples below.

A promoter can generally be characterized as either constitutive orinducible. Constitutive promoters are generally active or function todrive expression at all times (or at certain times in the cell lifecycle) at the same level. Inducible promoters, conversely, are active(or rendered inactive) or are significantly up- or down-regulated onlyin response to a stimulus. Both types of promoters find application inthe methods of the invention. Inducible promoters useful in theinvention include those that mediate transcription of an operably linkedgene in response to a stimulus, such as an exogenously provided smallmolecule (e.g, glucose, as in SEQ ID NO:1), temperature (heat or cold),lack of nitrogen in culture media, etc. Suitable promoters can activatetranscription of an essentially silent gene or upregulate, preferablysubstantially, transcription of an operably linked gene that istranscribed at a low level. Examples below describe additional induciblepromoters that are useful in Prototheca cells.

Inclusion of termination region control sequence is optional, and ifemployed, then the choice is be primarily one of convenience, as thetermination region is relatively interchangeable. The termination regionmay be native to the transcriptional initiation region (the promoter),may be native to the DNA sequence of interest, or may be obtainable fromanother source. See, for example, Chen and Orozco, Nucleic Acids Res.(1988) 16:8411.

The present invention also provides control sequences and recombinantgenes and vectors containing them that provide for the compartmentalizedexpression of a gene of interest. Organelles for targeting arechloroplasts, plastids, mitochondria, and endoplasmic reticulum. Inaddition, the present invention provides control sequences andrecombinant genes and vectors containing them that provide for thesecretion of a protein outside the cell.

Proteins expressed in the nuclear genome of Prototheca can be targetedto the plastid using plastid targeting signals. Plastid targetingsequences endogenous to Chlorella are known, such as genes in theChlorella nuclear genome that encode proteins that are targeted to theplastid; see for example GenBank Accession numbers AY646197 andAF499684, and in one embodiment, such control sequences are used in thevectors of the present invention to target expression of a protein to aPrototheca plastid.

The Examples below describe the use of algal plastid targeting sequencesto target heterologous proteins to the correct compartment in the hostcell. cDNA libraries were made using Prototheca moriformis and Chlorellaprotothecodies cells and are described in PCT Application No.PCT/US2009/066142.

In another embodiment of the present invention, the expression of apolypeptide in Prototheca is targeted to the endoplasmic reticulum. Theinclusion of an appropriate retention or sorting signal in an expressionvector ensure that proteins are retained in the endoplasmic reticulum(ER) and do not go downstream into Golgi. For example, theIMPACTVECTOR1.3 vector, from Wageningen UR-Plant Research International,includes the well known KDEL retention or sorting signal. With thisvector, ER retention has a practical advantage in that it has beenreported to improve expression levels 5-fold or more. The main reasonfor this appears to be that the ER contains lower concentrations and/ordifferent proteases responsible for post-translational degradation ofexpressed proteins than are present in the cytoplasm. ER retentionsignals functional in green microalgae are known. For example, see ProcNatl Acad Sci USA. 2005 Apr. 26; 102(17):6225-30.

In another embodiment of the present invention, a polypeptide istargeted for secretion outside the cell into the culture media. SeeHawkins et al., Current Microbiology Vol. 38 (1999), pp. 335-341 forexamples of secretion signals active in Chlorella that can be used, inaccordance with the methods of the invention, in Prototheca.

Many promoters are active in microalgae, including promoters that areendogenous to the algae being transformed, as well as promoters that arenot endogenous to the algae being transformed (i.e., promoters fromother algae, promoters from higher plants, and promoters from plantviruses or algae viruses). Exogenous and/or endogenous promoters thatare active in microalgae, and antibiotic resistance genes functional inmicroalgae are described by e.g., Curr Microbiol. 1997 December;35(6):356-62 (Chlorella vulgaris); Mar Biotechnol (NY). 2002 January;4(1):63-73 (Chlorella ellipsoidea); Mol Gen Genet. 1996 Oct. 16;252(5):572-9 (Phaeodactylum tricornutum); Plant Mol. Biol. 1996 April;31(1):1-12 (Volvox carteri); Proc Natl Acad Sci USA. 1994 Nov. 22;91(24):11562-6 (Volvox carteri); Falciatore A, Casotti R, Leblanc C,Abrescia C, Bowler C, PMID: 10383998, 1999 May; 1(3):239-251 (Laboratoryof Molecular Plant Biology, Stazione Zoologica, Villa Comunale, 1-80121Naples, Italy) (Phaeodactylum tricornutum and Thalassiosiraweissflogii); Plant Physiol. 2002 May; 129(1):7-12. (Porphyridium sp.);Proc Natl Acad Sci USA. 2003 Jan. 21; 100(2):438-42. (Chlamydomonasreinhardtii); Proc Natl Acad Sci USA. 1990 February; 87(3):1228-32.(Chlamydomonas reinhardtii); Nucleic Acids Res. 1992 Jun. 25;20(12):2959-65; Mar Biotechnol (NY). 2002 January; 4(1):63-73(Chlorella); Biochem Mol Biol Int. 1995 August; 36(5):1025-35(Chlamydomonas reinhardtii); J Microbiol. 2005 August; 43(4):361-5(Dunaliella); Yi Chuan Xue Bao. 2005 April; 32(4):424-33 (Dunaliella);Mar Biotechnol (NY). 1999 May; 1(3):239-251. (Thalassiosira andPhaedactylum); Koksharova, Appl Microbiol Biotechnol 2002 February;58(2):123-37 (various species); Mol Genet Genomics. 2004 February;271(1):50-9 (Thermosynechococcus elongates); J. Bacteriol. (2000), 182,211-215; FEMS Microbiol Lett. 2003 Apr. 25; 221(2):155-9; Plant Physiol.1994 June; 105(2):635-41; Plant Mol. Biol. 1995 December; 29(5):897-907(Synechococcus PCC 7942); Mar Pollut Bull. 2002; 45(1-12):163-7(Anabaena PCC 7120); Proc Natl Acad Sci USA. 1984 March; 81(5):1561-5(Anabaena (various strains)); Proc Natl Acad Sci USA. 2001 Mar. 27;98(7):4243-8 (Synechocystis); Wirth, Mol Gen Genet. 1989 March;216(1):175-7 (various species); Mol Microbiol, 2002 June; 44(6):1517-31and Plasmid, 1993 September; 30(2):90-105 (Fremyella diplosiphon); Hallet al. (1993) Gene 124: 75-81 (Chlamydomonas reinhardtii); Gruber et al.(1991). Current Micro. 22: 15-20; Jarvis et al. (1991) Current Genet.19: 317-322 (Chlorella); for additional promoters see also table 1 fromU.S. Pat. No. 6,027,900).

The promoter used to express an exogenous gene can be the promoternaturally linked to that gene or can be a heterologous gene. Somepromoters are active in more than one species of microalgae. Otherpromoters are species-specific. Preferred promoters include promoterssuch as RBCS2 from Chlamydomonas reinhardtii and viral promoters, suchas cauliflower mosaic virus (CMV) and chlorella virus, which have beenshown to be active in multiple species of microalgae (see for examplePlant Cell Rep. 2005 March; 23(10-11):727-35; J Microbiol. 2005 August;43(4):361-5; Mar Biotechnol (NY). 2002 January; 4(1):63-73). In otherembodiments, the Botryococcus malate dehydrogenase promoter, such anucleic acid comprising any part of SEQ ID NO: 150, or the Chlamydomonasreinhardtii RBCS2 promoter (SEQ ID NO: 151) can be used. Optionally, atleast 10, 20, 30, 40, 50, or 60 nucleotides or more of these sequencescontaining a promoter are used. Preferred promoters endogenous tospecies of the genus Chlorella are SEQ ID NO:1 and SEQ ID NO:2.

Preferred promoters useful for expression of exogenous genes inChlorella are listed in the sequence listing of this application, suchas the promoter of the Chlorella HUP1 gene (SEQ ID NO:1) and theChlorella ellipsoidea nitrate reductase promoter (SEQ ID NO:2).Chlorella virus promoters can also be used to express genes inChlorella, such as SEQ ID NOs: 1-7 of U.S. Pat. No. 6,395,965.Additional promoters active in Chlorella can be found, for example, inBiochem Biophys Res Commun. 1994 Oct. 14; 204(1):187-94; Plant Mol.Biol. 1994 October; 26(1):85-93; Virology. 2004 Aug. 15; 326(1):150-9;and Virology. 2004 Jan. 5; 318(1):214-23.

B. Genes and Codon Optimization

Typically, a gene includes a promoter, coding sequence, and terminationcontrol sequences. When assembled by recombinant DNA technology, a genemay be termed an expression cassette and may be flanked by restrictionsites for convenient insertion into a vector that is used to introducethe recombinant gene into a host cell. The expression cassette can beflanked by DNA sequences from the genome or other nucleic acid target tofacilitate stable integration of the expression cassette into the genomeby homologous recombination. Alternatively, the vector and itsexpression cassette may remain unintegrated, in which case, the vectortypically includes an origin of replication, which is capable ofproviding for replication of the heterologous vector DNA.

A common gene present on a vector is a gene that codes for a protein,the expression of which allows the recombinant cell containing theprotein to be differentiated from cells that do not express the protein.Such a gene, and its corresponding gene product, is called a selectablemarker. Any of a wide variety of selectable markers can be employed in atransgene construct useful for transforming Prototheca. Examples ofsuitable selectable markers include the G418 resistance gene, thenitrate reductase gene (see Dawson et al. (1997), Current Microbiology35:356-362), the hygromycin phosphotransferase gene (HPT; see Kim et al.(2002), Mar. Biotechnol. 4:63-73), the neomycin phosphotransferase gene,the ble gene, which confers resistance to phleomycin (Huang et al.(2007), Appl. Microbiol. Biotechnol. 72:197-205), and theaminoglycoside-3′-O-phosphotransferase (SEQ ID NO: 194), which confersresistance to kanamycin. Methods of determining sensitivity ofmicroalgae to antibiotics are well known. For example, Mol Gen Genet.1996 Oct. 16; 252(5):572-9.

Other selectable markers that are not antibiotic-based can also beemployed in a transgene construct useful for transforming microalgae ingeneral, including Prototheca species. Genes that confers the ability toutilize certain carbon sources that were previously unable to beutilized by the microalgae can also be used as a selectable marker. Byway of illustration, Prototheca moriformis strains typically growpoorly, if at all, on sucrose. Using a construct containing a sucroseinvertase gene can confer the ability of positive transformants to growon sucrose as a carbon substrate. Additional details on using sucroseutilization as a selectable marker along with other selectable markersare discussed in Section IV below.

For purposes of the present invention, the expression vector used toprepare a recombinant host cell of the invention will include at leasttwo, and often three, genes, if one of the genes is a selectable marker.For example, a genetically engineered Prototheca of the invention can bemade by transformation with vectors of the invention that comprise, inaddition to a selectable marker, one or more exogenous genes, such as,for example, sucrose invertase gene or acyl ACP-thioesterase gene. Oneor both genes can be expressed using an inducible promoter, which allowsthe relative timing of expression of these genes to be controlled toenhance the lipid yield and conversion to fatty acid esters. Expressionof the two or more exogenous genes may be under control of the sameinducible promoter or under control of different inducible (orconstitutive) promoters. In the latter situation, expression of a firstexogenous gene can be induced for a first period of time (during whichexpression of a second exogenous gene may or may not be induced) andexpression of a second exogenous gene can be induced for a second periodof time (during which expression of a first exogenous gene may or maynot be induced).

In other embodiments, the two or more exogenous genes (in addition toany selectable marker) are: a fatty acyl-ACP thioesterase and a fattyacyl-CoA/aldehyde reductase, the combined action of which yields analcohol product. Further provided are other combinations of exogenousgenes, including without limitation, a fatty acyl-ACP thioesterase and afatty acyl-CoA reductase to generate aldehydes. In one embodiment, thevector provides for the combination of a fatty acyl-ACP thioesterase, afatty acyl-CoA reductase, and a fatty aldehyde decarbonylase to generatealkanes. In each of these embodiments, one or more of the exogenousgenes can be expressed using an inducible promoter.

Other illustrative vectors of the invention that express two or moreexogenous genes include those encoding both a sucrose transporter and asucrose invertase enzyme and those encoding both a selectable marker anda secreted sucrose invertase. The recombinant Prototheca transformedwith either type of vector produce lipids at lower manufacturing costdue to the engineered ability to use sugar cane (and sugar cane-derivedsugars) as a carbon source. Insertion of the two exogenous genesdescribed above can be combined with the disruption of polysaccharidebiosynthesis through directed and/or random mutagenesis, which steersever greater carbon flux into lipid production. Individually and incombination, trophic conversion, engineering to alter lipid productionand treatment with exogenous enzymes alter the lipid compositionproduced by a microorganism. The alteration can be a change in theamount of lipids produced, the amount of one or more hydrocarbon speciesproduced relative to other lipids, and/or the types of lipid speciesproduced in the microorganism. For example, microalgae can be engineeredto produce a higher amount and/or percentage of TAGs.

For optimal expression of a recombinant protein, it is beneficial toemploy coding sequences that produce mRNA with codons preferentiallyused by the host cell to be transformed. Thus, proper expression oftransgenes can require that the codon usage of the transgene matches thespecific codon bias of the organism in which the transgene is beingexpressed. The precise mechanisms underlying this effect are many, butinclude the proper balancing of available aminoacylated tRNA pools withproteins being synthesized in the cell, coupled with more efficienttranslation of the transgenic messenger RNA (mRNA) when this need ismet. When codon usage in the transgene is not optimized, available tRNApools are not sufficient to allow for efficient translation of theheterologous mRNA resulting in ribosomal stalling and termination andpossible instability of the transgenic mRNA.

The present invention provides codon-optimized nucleic acids useful forthe successful expression of recombinant proteins in Prototheca. Codonusage in Prototheca species was analyzed by studying cDNA sequencesisolated from Prototheca moriformis. This analysis represents theinterrogation over 24,000 codons and resulted in Table 2 below.

TABLE 2 Preferred codon usage in Prototheca strains. Ala GCG 345 (0.36)Asn AAT 8 (0.04) GCA 66 (0.07) AAC 201 (0.96) GCT 101 (0.11) Pro CCG 161(0.29) GCC 442 (0.46) CCA 49 (0.09) Cys TGT 12 (0.10) CCT 71 (0.13) TGC105 (0.90) CCC 267 (0.49) Asp GAT 43 (0.12) Gln CAG 226 (0.82) GAC 316(0.88) CAA 48 (0.18) Glu GAG 377 (0.96) Arg AGG 33 (0.06) GAA 14 (0.04)AGA 14 (0.02) Phe TTT 89 (0.29) CGG 102 (0.18) TTC 216 (0.71) CGA 49(0.08) Gly GGG 92 (0.12) CGT 51 (0.09) GGA 56 (0.07) CGC 331 (0.57) GGT76 (0.10) Ser AGT 16 (0.03) GGC 559 (0.71) AGC 123 (0.22) His CAT 42(0.21) TCG 152 (0.28) CAC 154 (0.79) TCA 31 (0.06) Ile ATA 4 (0.01) TCT55 (0.10) ATT 30 (0.08) TCC 173 (0.31) ATC 338 (0.91) Thr ACG 184 (0.38)Lys AAG 284 (0.98) ACA 24 (0.05) AAA 7 (0.02) ACT 21 (0.05) Leu TTG 26(0.04) ACC 249 (0.52) TTA 3 (0.00) Val GTG 308 (0.50) CTG 447 (0.61) GTA9 (0.01) CTA 20 (0.03) GTT 35 (0.06) CTT 45 (0.06) GTC 262 (0.43) CTC190 (0.26) Trp TGG 107 (1.00) Met ATG 191 (1.00) Tyr TAT 10 (0.05) TAC180 (0.95) Stop TGA/TAG/TAA

In other embodiments, the gene in the recombinant vector has beencodon-optimized with reference to a microalgal strain other than aPrototheca strain. For example, methods of recoding genes for expressionin microalgae are described in U.S. Pat. No. 7,135,290. Additionalinformation for codon optimization is available, e.g., at the codonusage database of GenBank.

Other non-limiting examples of codon usage in Chlorella pyrenoidosa,Dunaliella salina, and Chlorella protothecoides are shown in Tables 2A,2B, and 2C, respectively.

TABLE 2A Codon usage in Chlorella pyrenoidosa. Phe UUU 39 (0.82) Ser UCU50 (1.04) UUC 56 (1.18) UCC 60 (1.25) Leu UUA 10 (0.20) UCA 6 (0.96) UUG46 (0.91) UCG 43 (0.89) Tyr UAU 15 (0.59) Cys UGU 46 (0.77) UAC 36(1.41) UGC 73 (1.23) ter UAA 9 (0.00) ter UGA 43 (0.00) ter UAG 15(0.00) Trp UGG 69 (1.00) Leu CUU 49 (0.97) Pro CCU 80 (0.98) CUC 73(1.45) CCC 88 (1.08) CUA 22 (0.44) CCA 93 (1.14) CUG 103 (2.04) CCG 65(0.80) His CAU 50 (0.88) Arg CGU 39 (0.76) CAC 3 (1.12) CGC 63 (1.23)Gln CAA 59 (0.84) CGA 46 (0.90) CAG 2 (1.16) CGG 47 (0.92) Ile AUU 24(0.69) Thr ACU 32 (0.67) AUC 61 (1.76) ACC 76 (1.60) AUA 19 (0.55) ACA41 (0.86) Met AUG 42 (1.00) ACG 41 (0.86) Asn AAU 26 (0.75) Ser AGU 23(0.48) AAC 3 (1.25) AGC 67 (1.39) Lys AAA 32 (0.54) Arg AGA 51 (1.00)AAG 86 (1.46) AGG 61 (1.19) Val GUU 36 (0.75) Ala GCU 57 (0.79) GUC 54(1.13) GCC 97 (1.34) GUA 30 (0.63) GCA 89 (1.23) GUG 71 (1.49) GCG 47(0.65) Asp GAU 60 (0.95) Gly GGU 35 (0.60) GAC 66 (1.05) GGC 78 (1.33)Glu GAA 41 (0.68) GGA 54 (0.92) GAG 80 (1.32) GGG 67 (1.15)

TABLE 2B Preferred codon usage in Dunaliella salina. TTC (Phe) TAC (Tyr)TGC (Cys) TAA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg) CTG (Leu)CAG (Gln) ATC (Ile) ACC (Thr) AAC (Asn) AGC (Ser) ATG (Met) AAG (Lys)GCC (Ala) GAC (Asp) GGC (Gly) GTG (Val) GAG (Glu)

TABLE 2C Preferred codon usage in Chlorella protothecoides. TTC (Phe)TAC (Tyr) TGC (Cys) TGA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg)CTG (Leu) CAG (Gln) ATC (Ile) ACC (Thr) GAC (Asp) TCC (Ser) ATG (Met)AAG (Lys) GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val) GAG (Glu)

C. Inducible Expression

The present invention also provides for the use of an inducible promoterto express a gene of interest. In particular, the use of an induciblepromoter to express a lipase gene permits production of the lipase aftergrowth of the microorganism when conditions have been adjusted, ifnecessary, to enhance transesterification, for example, after disruptionof the cells, reduction of the water content of the reaction mixture,and/or addition sufficient alcohol to drive conversion of TAGs to fattyacid esters.

Inducible promoters useful in the invention include those that mediatetranscription of an operably linked gene in response to a stimulus, suchas an exogenously provided small molecule (e.g, glucose, as in SEQ IDNO:1), temperature (heat or cold), light, etc. Suitable promoters canactivate transcription of an essentially silent gene or upregulate,preferably substantially, transcription of an operably linked gene thatis transcribed at a low level. In the latter case, the level oftranscription of the lipase preferably does not significantly interferewith the growth of the microorganism in which it is expressed.

Expression of transgenes in Chlorella can be performed inducibly throughpromoters such as the promoter that drives the Chlorella hexosetransporter gene (SEQ ID NO:1). This promoter is strongly activated bythe presence of glucose in the culture media.

D. Expression of Two or More Exogenous Genes

Further, a genetically engineered microorganism, such as a microalgae,may comprise and express two or more exogenous genes, such as, forexample, a lipase and a lytic gene, e.g., one encoding apolysaccharide-degrading enzyme. One or both genes can be expressedusing an inducible promoter, which allows the relative timing ofexpression of these genes to be controlled to enhance the lipid yieldand conversion to fatty acid esters. Expression of the two or moreexogenous genes may be under control of the same inducible promoter orunder control of a different inducible promoters. In the lattersituation, expression of a first exogenous gene can be induced for afirst period of time (during which expression of a second exogenous genemay or may not be induced) and expression of a second exogenous gene canbe induced for a second period of time (during which expression of afirst exogenous gene may or may not be induced). Provided herein arevectors and methods for engineering lipid-producing microbes tometabolize sucrose, which is an advantageous trait because it allows theengineered cells to convert sugar cane feedstocks into lipids.

Also provided herein are genetically engineered strains of microbes(e.g., microalgae, oleaginous yeast, bacteria, or fungi) that expresstwo or more exogenous genes, such as, for example, a fatty acyl-ACPthioesterase and a fatty acyl-CoA/aldehyde reductase, the combinedaction of which yields an alcohol product. Further provided are othercombinations of exogenous genes, including without limitation, a fattyacyl-ACP thioesterase and a fatty acyl-CoA reductase to generatealdehydes. In addition, this application provides for the combination ofa fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, and a fattyaldehyde decarbonylase to generate alkanes. One or more of the exogenousgenes can be expressed using an inducible promoter.

Examples of further modifications suitable for use in the presentinvention are include genetically engineering strains of microalgae toexpress two or more exogenous genes, one encoding a transporter of afixed carbon source (such as sucrose) and a second encoding a sucroseinvertase enzyme. The resulting fermentable organisms producehydrocarbons at lower manufacturing cost than what has been obtainableby previously known methods of biological hydrocarbon production.Insertion of the two exogenous genes described above can be combinedwith the disruption of polysaccharide biosynthesis through directedand/or random mutagenesis, which steers ever greater carbon flux intohydrocarbon production. Individually and in combination, trophicconversion, engineering to alter hydrocarbon production and treatmentwith exogenous enzymes alter the hydrocarbon composition produced by amicroorganism. The alteration can be a change in the amount ofhydrocarbons produced, the amount of one or more hydrocarbon speciesproduced relative to other hydrocarbons, and/or the types of hydrocarbonspecies produced in the microorganism. For example, microalgae can beengineered to produce a higher amount and/or percentage of TAGs.

E. Compartmentalized Expression

The present invention also provides for compartmentalized expression ofa gene of interest. In particular, it can be advantageous, in particularembodiments, to target expression of the lipase to one or more cellularcompartments, where it is sequestered from the majority of cellularlipids until initiation of the transesterification reaction. Preferredorganelles for targeting are chloroplasts, mitochondria, and endoplasmicreticulum.

(1) Expression in Chloroplasts

In one embodiment of the present invention, the expression of apolypeptide in a microorganism is targeted to chloroplasts. Methods fortargeting expression of a heterologous gene to the chloroplast are knownand can be employed in the present invention. Methods for targetingforeign gene products into chloroplasts are described in Shrier et al.,EMBO J. (1985) 4:25 32. See also Tomai et al. Gen. Biol. Chem. (1988)263:15104 15109 and U.S. Pat. No. 4,940,835 for the use of transitpeptides for translocating nuclear gene products into the chloroplast.Methods for directing the transport of proteins to the chloroplast arealso reviewed in Kenauf TIBTECH (1987) 5:40 47. Chloroplast targetingsequences endogenous to Chlorella are known, such as genes in theChlorella nuclear genome that encode proteins that are targeted to thechloroplast; see for example GenBank Accession numbers AY646197 andAF499684.

Wageningen UR-Plant Research International sells an IMPACTVECTOR1.4vector, which uses the secretion signal of the Chrysanthemum morifoliumsmall subunit protein to deliver a heterologous protein into thechloroplast stroma (cytoplasmic) environment, shuttling across a doublemembrane system. The protein is fused to the first 11 amino acids of themature rubisco protein in order to allow proper processing of the signalpeptide (Wong et al., Plant Molecular Biology 20: 81-93 (1992)). Thesignal peptide contains a natural intron from the RbcS gene.

In another approach, the chloroplast genome is genetically engineered toexpress the heterologous protein. Stable transformation of chloroplastsof Chlamydomonas reinhardtii (a green alga) using bombardment ofrecipient cells with high-velocity tungsten microprojectiles coated withforeign DNA has been described. See, for example, Boynton et al.,Science (1988) 240: 1534 1538; Blowers et al. Plant Cell (1989) 1:123132 and Debuchy et al., EMBO J. (1989) δ: 2803 2809. The transformationtechnique, using tungsten microprojectiles, is described by Klein etal., Nature (London) (1987) 7:70 73. Other methods of chloroplasttransformation for both plants and microalgae are known. See for exampleU.S. Pat. Nos. 5,693,507; 6,680,426; and Plant Physiol. 2002 May;129(1):7-12; and Plant Biotechnol J. 2007 May; 5(3):402-12.

As described in U.S. Pat. No. 6,320,101 (issued Nov. 20, 2001 to Kaplanet al.; which is incorporated herein by reference), cells can bechemically treated so as to reduce the number of chloroplasts per cellto about one. Then, the heterologous nucleic acid can be introduced intothe cells via particle bombardment with the aim of introducing at leastone heterologous nucleic acid molecule into the chloroplasts. Theheterologous nucleic acid is selected such that it is integratable intothe chloroplast's genome via homologous recombination which is readilyeffected by enzymes inherent to the chloroplast. To this end, theheterologous nucleic acid includes, in addition to a gene of interest,at least one nucleic acid sequence that is derived from thechloroplast's genome. In addition, the heterologous nucleic acidtypically includes a selectable marker. Further details relating to thistechnique are found in U.S. Pat. Nos. 4,945,050 and 5,693,507 which areincorporated herein by reference. A polypeptide can thus be produced bythe protein expression system of the chloroplast.

U.S. Pat. No. 7,135,620 (issued Nov. 14, 2006 to Daniell et al.;incorporated herein by reference) describes chloroplast expressionvectors and related methods. Expression cassettes are DNA constructsincluding a coding sequence and appropriate control sequences to providefor proper expression of the coding sequence in the chloroplast. Typicalexpression cassettes include the following components: the 5′untranslated region from a microorganism gene or chloroplast gene suchas psbA which will provide for transcription and translation of a DNAsequence encoding a polypeptide of interest in the chloroplast; a DNAsequence encoding a polypeptide of interest; and a translational andtranscriptional termination region, such as a 3′ inverted repeat regionof a chloroplast gene that can stabilize RNA of introduced genes,thereby enhancing foreign gene expression. The cassette can optionallyinclude an antibiotic resistance gene.

Typically, the expression cassette is flanked by convenient restrictionsites for insertion into an appropriate genome. The expression cassettecan be flanked by DNA sequences from chloroplast DNA to facilitatestable integration of the expression cassette into the chloroplastgenome, particularly by homologous recombination. Alternatively, theexpression cassette may remain unintegrated, in which case, theexpression cassette typically includes a chloroplast origin ofreplication, which is capable of providing for replication of theheterologous DNA in the chloroplast.

The expression cassette generally includes a promoter region from a genecapable of expression in the chloroplast. The promoter region mayinclude promoters obtainable from chloroplast genes, such as the psbAgene from spinach or pea, or the rbcL and atpB promoter region frommaize and Rrna promoters. Examples of promoters are described inHanley-Bowdoin and Chua, TIBS (1987) 12:67 70; Mullet et al., PlantMolec Biol. (1985) 4: 39 54; Hanley-Bowdoin (1986) PhD. Dissertation,the Rockefeller University; Krebbers et al., Nucleic Acids Res. (1982)10: 4985 5002; Zurawaki et al., Nucleic Acids Res. (1981) 9:3251 3270;and Zurawski et al., Proc. Nat'l Acad. Sci. U.S.A. (1982) 79: 7699 7703.Other promoters can be identified and the relative strength of promotersso identified evaluated, by placing a promoter of interest 5′ to apromoterless marker gene and observing its effectiveness relative totranscription obtained from, for example, the promoter from the psbAgene, a relatively strong chloroplast promoter. The efficiency ofheterologus gene expression additionally can be enhanced by any of avariety of techniques. These include the use of multiple promotersinserted in tandem 5′ to the heterologous gente, for example a doublepsbA promoter, the addition of enhancer sequences and the like.

Numerous promoters active in the Chlorella chloroplast can be used forexpression of exogenous genes in the Chlorella chloroplast, such asthose found in GenBank accession number NC_(—)001865 (Chlorella vulgarischloroplast, complete genome),

Where it is desired to provide for inducible expression of theheterologous gene, an inducible promoter and/or a 5′ untranslated regioncontaining sequences which provide for regulation at the level oftranscription and/or translation (at the 3′ end) may be included in theexpression cassette. For example, the 5′ untranslated region can be froma gene wherein expression is regulatable by light. Similarly, 3′inverted repeat regions could be used to stabilize RNA of heterologousgenes. Inducible genes may be identified by enhanced expression inresponse to a particular stimulus of interest and low or absentexpression in the absence of the stimulus. For example, alight-inducible gene can be identified where enhanced expression occursduring irradiation with light, while substantially reduced expression orno expression occurs in low or no light. Light regulated promoters fromgreen microalgae are known (see for example Mol Genet Genomics. 2005December; 274(6):625-36).

The termination region which is employed will be primarily one ofconvenience, since the termination region appears to be relativelyinterchangeable among chloroplasts and bacteria. The termination regionmay be native to the transcriptional initiation region, may be native tothe DNA sequence of interest, or may be obtainable from another source.See, for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:8411.

The expression cassettes may be transformed into a plant cell ofinterest by any of a number of methods. These methods include, forexample, biolistic methods (See, for example, Sanford, Trends InBiotech. (1988) 6:299 302, U.S. Pat. No. 4,945,050; electroporation(Fromm et al., Proc. Nat'l. Acad. Sci. (USA) (1985) 82:5824 5828); useof a laser beam, microinjection or any other method capable ofintroducing DNA into a chloroplast.

Additional descriptions of chloroplast expression vectors suitable foruse in microorganisms such as microalgae are found in U.S. Pat. No.7,081,567 (issued Jul. 25, 2006 to Xue et al.); U.S. Pat. No. 6,680,426(issued Jan. 20, 2004 to Daniell et al.); and U.S. Pat. No. 5,693,507(issued Dec. 2, 1997 to Daniell et al.).

Proteins expressed in the nuclear genome of Chlorella can be targeted tothe chloroplast using chloroplast targeting signals. Chloroplasttargeting sequences endogenous to Chlorella are known, such as genes inthe Chlorella nuclear genome that encode proteins that are targeted tothe chloroplast; see for example GenBank Accession numbers AY646197 andAF499684. Proteins can also be expressed in the Chlorella chloroplast byinsertion of genes directly into the chloroplast genome. Chloroplasttransformation typically occurs through homologous recombination, andcan be performed if chloroplast genome sequences are known for creationof targeting vectors (see for example the complete genome sequence of aChlorella chloroplast; Genbank accession number NC_(—)001865). Seeprevious sections herein for details of chloroplast transformation.

(2) Expression in Mitochondria

In another embodiment of the present invention, the expression of apolypeptide in a microorganism is targeted to mitochondria. Methods fortargeting foreign gene products into mitochnodria (Boutry et al. Nature(London) (1987) 328:340 342) have been described, including in greenmicroalgae (see for example Mol Gen Genet. 1993 January;236(2-3):235-44).

For example, an expression vector encoding a suitable secretion signalcan target a heterologus protein to the mitochondrion. TheIMPACTVECTOR1.5 vector, from Wageningen UR-Plant Research International,uses the yeast CoxIV secretion signal, which was shown to deliverproteins in the mitochondrial matrix. The protein is fused to the first4 amino acids of the yeast CoxIV protein in order to allow properprocessing of the signal peptide (Kohler et al. Plant J 11: 613-621(1997)). Other mitochondrial targeting sequences are known, includingthose functional in green microalgae. For example, see FEBS Lett. 1990Jan. 29; 260(2):165-8; and J Biol. Chem. 2002 Feb. 22; 277(8):6051-8.

Proteins expressed in the nuclear genome of Chlorella can be targeted tothe mitochondria using mitochondrial targeting signals. See previoussections herein for details of mitochondrial protein targeting andtransformation.

(3) Expression in Endoplasmic Reticulum

In another embodiment of the present invention, the expression of apolypeptide in a microorganism is targeted to the endoplasmic reticulum.The inclusion of an appropriate retention or sorting signal in anexpression vector ensure that proteins are retained in the endoplasmicreticulum (ER) and do not go downstream into Golgi. For example, theIMPACTVECTOR1.3 vector, from Wageningen UR-Plant Research International,includes the well known KDEL retention or sorting signal. With thisvector, ER retention has a practical advantage in that it has beenreported to improve expression levels 5-fold or more. The main reasonfor this appears to be that the ER contains lower concentrations and/ordifferent proteases responsible for post-translational degradation ofexpressed proteins than are present in the cytoplasm. ER retentionsignals functional in green microalgae are known. For example, see ProcNatl Acad Sci USA. 2005 Apr. 26; 102(17):6225-30.

While the methods and materials of the invention allow for theintroduction of any exogenous gene into a microorganism, for examplePrototheca, genes relating to sucrose utilization and lipid pathwaymodification are of particular interest, as discussed in the followingsections.

IV. Selectable Markers

1. Sucrose Utilization

In embodiment, the recombinant Prototheca cell of the invention furthercontains one or more exogenous sucrose utilization genes. In variousembodiments, the one or more genes encode one or more proteins selectedfrom the group consisting of a fructokinase, a glucokinase, ahexokinase, a sucrose invertase, a sucrose transporter. For example,expression of a sucrose transporter and a sucrose invertase allowsPrototheca to transport sucrose into the cell from the culture media andhydrolyze sucrose to yield glucose and fructose. Optionally, afructokinase can be expressed as well in instances where endogenoushexokinase activity is insufficient for maximum phosphorylation offructose. Examples of suitable sucrose transporters are Genbankaccession numbers CAD91334, CAB92307, and CAA53390. Examples of suitablefructokinases are Genbank accession numbers P26984, P26420 and CAA43322.

In one embodiment, the present invention provides a Prototheca host cellthat secretes a sucrose invertase. Secretion of a sucrose invertaseobviates the need for expression of a transporter that can transportsucrose into the cell. This is because a secreted invertase catalyzesthe conversion of a molecule of sucrose into a molecule of glucose and amolecule of fructose, both of which can be transported and utilized bymicrobes provided by the invention. For example, expression of a sucroseinvertase (such as SEQ ID NO:3) with a secretion signal (such as that ofSEQ ID NO: 4 (from yeast), SEQ ID NO: 5 (from higher plants), SEQ ID NO:6 (eukaryotic consensus secretion signal), and SEQ ID NO: 7 (combinationof signal sequence from higher plants and eukaryotic consensus)generates invertase activity outside the cell. Expression of such aprotein, as enabled by the genetic engineering methodology disclosedherein, allows cells already capable of utilizing extracellular glucoseas an energy source to utilize sucrose as an extracellular energysource.

Prototheca species expressing an invertase in media containing sucroseare a preferred microalgal species for the production of oil. Theexpression and extracellular targeting of this fully active proteinallows the resulting host cells to grow on sucrose, whereas theirnon-transformed counterparts cannot. Thus, the present inventionprovides Prototheca recombinant cells with a codon-optimized invertasegene, including but not limited to the yeast invertase gene, integratedinto their genome such that the invertase gene is expressed as assessedby invertase activity and sucrose hydrolysis. The present invention alsoprovides invertase genes useful as selectable markers in Protothecarecombinant cells, as such cells are able to grow on sucrose, whiletheir non-transformed counterparts cannot; and methods for selectingrecombinant host cells using an invertase as a powerful, selectablemarker for algal molecular genetics.

The successful expression of a sucrose invertase in Prototheca alsoillustrates another aspect of the present invention in that itdemonstrates that heterologous (recombinant) proteins can be expressedin the algal cell and successfully transit outside of the cell and intothe culture medium in a fully active and functional form. Thus, thepresent invention provides methods and reagents for expressing a wideand diverse array of heterologous proteins in microalgae and secretingthem outside of the host cell. Such proteins include, for example,industrial enzymes such as, for example, lipases, proteases, cellulases,pectinases, amylases (e.g., SEQ ID NO: 190-191), esterases,oxidoreductases, transferases, lactases, isomerases, and invertases, aswell as therapeutic proteins such as, for example, growth factors,cytokines, full length antibodies comprising two light and two heavychains, Fabs, scFvs (single chain variable fragment), camellid-typeantibodies, antibody fragments, antibody fragment-fusions,antibody-receptor fusions, insulin, interferons, and insulin-like growthfactors.

The successful expression of a sucrose invertase in Prototheca alsoillustrates another aspect of the present invention in that it providesmethods and reagents for the use of fungal transit peptides in algae todirect secretion of proteins in Prototheca; and methods and reagents fordetermining if a peptide can function, and the ability of it tofunction, as a transit peptide in Prototheca cells. The methods andreagents of the invention can be used as a tool and platform to identifyother transit peptides that can successfully traffic proteins outside ofa cell, and that the yeast invertase has great utility in these methods.As demonstrated in this example, removal of the endogenous yeastinvertase transit peptide and its replacement by other transit peptides,either endogenous to the host algae or from other sources (eukaryotic,prokaryotic and viral), can identify whether any peptide of interest canfunction as a transit peptide in guiding protein egress from the cell.

Examples of suitable sucrose invertases include those identified byGenbank accession numbers CAB95010, NP_(—)012104 and CAA06839.Non-limiting examples of suitable invertases are listed below in Table 3Amino acid sequences for each listed invertase are included in theSequence Listing below. In some cases, the exogenous sucrose utilizationgene suitable for use in the methods and vectors of the inventionencodes a sucrose invertase that has at least 40, 50, 60, 75, or 90% orhigher amino acid identity with a sucrose invertase selected from Table3.

TABLE 3 Sucrose invertases. GenBank Description Organism Accession No.SEQ ID NO: Invertase Chicorium intybus Y11124 SEQ ID NO: 20 InvertaseSchizosaccharomyces AB011433 SEQ ID NO: 21 pombe beta-fructofuranosidasePichia anomala X80640 SEQ ID NO: 22 (invertase) Invertase Debaryomycesoccidentalis X17604 SEQ ID NO: 23 Invertase Oryza sativa AF019113 SEQ IDNO: 24 Invertase Allium cepa AJ006067 SEQ ID NO: 25 Invertase Betavulgaris subsp. AJ278531 SEQ ID NO: 26 Vulgaris beta-fructofuranosidaseBifidobacterium breve AAT28190 SEQ ID NO: 27 (invertase) UCC2003Invertase Saccharomyces cerevisiae NP_012104 SEQ ID NO: 8 (nucleotide)SEQ ID NO: 28 (amino acid) Invertase A Zymomonas mobilis AAO38865 SEQ IDNO: 29 Invertase Arabadopsis NP_566464 SEQ ID NO: 188 thaliana

The secretion of an invertase to the culture medium by Prototheca enablethe cells to grow as well on waste molasses from sugar cane processingas they do on pure reagent-grade glucose; the use of this low-valuewaste product of sugar cane processing can provide significant costsavings in the production of lipids and other oils. Thus, the presentinvention provides a microbial culture containing a population ofPrototheca microorganisms, and a culture medium comprising (i) sucroseand (ii) a sucrose invertase enzyme. In various embodiments the sucrosein the culture comes from sorghum, sugar beet, sugar cane, molasses, ordepolymerized cellulosic material (which may optionally contain lignin).In another aspect, the methods and reagents of the inventionsignificantly increase the number and type of feedstocks that can beutilized by recombinant Prototheca. While the microbes exemplified hereare altered such that they can utilize sucrose, the methods and reagentsof the invention can be applied so that feedstocks such as cellulosicsare utilizable by an engineered host microbe of the invention with theability to secrete cellulases, pectinases, isomerases, or the like, suchthat the breakdown products of the enzymatic reactions are no longerjust simply tolerated but rather utilized as a carbon source by thehost. An example of this is described below and in the Examples ofmicrobes engineered to express a secretable α-galactosidase, conferringthe ability to hydrolyze α-galactosyl bonds in oligosaccharides such asthose contained in raffinose and stachyose which are twooligosaccharides found in agricultural waste streams.

2. Alpha-galactosidase Expression

While the expression of a sucrose invertase, as described above, confersthe ability for Prototheca cells to more efficiently utilize sucrose asa carbon source (via the enzyme hydrolyzing the α-linkage betweenfructose and glucose molecules in the disaccharide sucrose), theexpression of other enzymes that hydrolyze other types of α-linkages inoligosaccharides can confer the ability for Prototheca cells to utilizeother carbon sources. The expression of these enzymes (and the resultingability to utilize carbon sources that Prototheca and other microalgalcells ordinarily would not be able to) can be used as a selectablemarker for these transgenic Prototheca cells by allowing for theselection of positive clones that are able to grow on these carbonsources.

In an embodiment, the recombinant Prototheca cell of the inventionfurther contains one or more exogenous genes encodingpolysaccharide-degrading enzymes. In various embodiments, the one ormore genes encoding a polysaccharide-degrading enzyme is a gene encodinga secreted α-galactosidase. The expression of an exogenous secretedα-galactosidase in a Prototheca cell confers the ability of suchtransformed strains to grow on sugars (carbon sources) containingD-galactosyl linkages, such as α-linkages between galactose and glucosemonosaccharide units. Prototheca strains expressing an exogenous,secreted α-galactosidase will be able to utilize disaccharides such asmelibiose (disaccharide composed of α-D-galactose-glucose).

Sugars such as raffinose (a trisaccharide comprised of α-linkedgalactose-glucose-fructose) and stachyose (a tetrasaccharide composed totwo α-linked D-galactose units, followed by α-linked glucose andfructose) are present in significant proportions in agricultural wastestreams such as beet pulp (raffinose) and soybean meal (stachyose). Suchagricultural residues represent a significant untapped carbon source forthe conversion into oil by microbes (including Prototheca) capable ofutilizing them.

Prototheca strains are unable to utilize oligosaccharides such asraffinose and stachyose in any significant quantity or at all. In thecase of raffinose and stachyose, although transgenic strains expressinga sucrose invertase (as described above) have the ability to hydrolyzethe α-linkage between fructose and glucose in α-galactosyl derivativesof sucrose, but the remainder of the oligosaccharide remains unutilized,as sucrose invertase will not cleave the remaining α-linkages in suchsugars and the resulting disaccharides are not utilizable. In anotherembodiment, the recombinant Prototheca cell of the invention comprisesboth an exogenous gene encoding a sucrose invertase and an exogenousgene encoding an α-galactosidase. Thus, strains expressing both asucrose invertase and an α-galactosidase will be capable of fullyhydrolyzing oligosaccharides such as raffinose and stachyose, enablingthe consumption of the component monomers. In addition, α-galactosidaseencoding genes may be used as a selectable marker for transformation.Clones containing the exogenous α-galactosidase gene will have theability to grow on melibiose. Examples of suitable α-galactosidase genesfor use in Prototheca strains include the MEL1 gene from Saccharomycescarlbergensis, the AglC gene from Aspergilus niger. Interestingly, notall α-galactosidase genes are functional in Prototheca species, even ifthe genes are optimized according to the preferred codon usage inPrototheca strains. The Examples below demonstrates the ability oftransgenic Prototheca cells to grow on melibiose when transformed withcodon-optimized MEL1 gene from S. carlbergensis and the AglC gene fromA. niger, but not an α-galactosidase encoding gene from the higherplant, Cyamopsis tetragonobola (Guar bean).

3. Thiamine Auxotrophy Complementation

Prototheca strains including Prototheca moriformis are known to bethiamine auxotrophic (See, for example, Ciferri, O. (1956) Nature, v.178, pp. 1475-1476), meaning that these strains require thiamine in thenutrient media for growth Thiamine auxotrophy can be the result ofmutations or lack of expression of enzymes in the thiamine biosyntheticpathway. Complemented transgenic strains expressing the missingenzyme(s) in the thiamine biosynthetic pathway can then be grown withoutadded thiamine, thus reducing the cost of the nutrient media as well asrendering the resulting microalgal biomass more desirable from an animalnutrition perspective. Complementation with a thiamine biosyntheticpathway enzyme can also be used as a selectable marker as the transgenicgene confers the ability to grow on plates/media that does not containthiamine.

In an embodiment, the recombinant Prototheca cell of the inventionfurther contains one or more exogenous genes encoding thiaminebiosynthetic pathway enzyme. In another embodiment, the recombinantPrototheca cell of the invention comprises an exogenous gene encodinghydroxymethylpyrimidine phosphate synthases (e.g., SEQ ID NO: 192) fromalgal, plant or cyanobacterial sources. In still other embodiments, thehydroxymethylpyrimidine phosphate synthase is encoded by a THIC gene. Instill other embodiments, the THIC gene is the Coccomyxa C-169 THIC,Arabidopsis thaliana THIC, the Synechocystis sp. PCC 6803 THIC, or theSalmonella enterica subsp. enterica serovar Typhimurium str.THIC (SEQ IDNO: 193). The Examples below details the engineering of Protothecamoriformis UTEX 1435 with restored thiamine prototrophy.

4. Other Selectable Markers

Any of a wide variety of selectable markers can be employed in atransgene construct useful for transforming microorganisms, such asChlorella. Examples of suitable selectable markers include the nitratereductase gene, the hygromycin phosphotransferase gene (HPT), theneomycin phosphotransferase gene, and the ble gene, which confersresistance to phleomycin. Methods of determining sensitivity ofmicroalgae to antibiotics are well known. For example, Mol Gen Genet.1996 Oct. 16; 252(5):572-9.

More specifically, Dawson et al. (1997), Current Microbiology 35:356-362(incorporated by reference herein in its entirety), described the use ofthe nitrate reductase (NR) gene from Chlorella vulgaris as a selectablemarker for NR-deficient Chlorella sorokiniana mutants. Kim et al.(2002), Mar. Biotechnol. 4:63-73 (incorporated by reference herein inits entirety), disclosed the use of the HPT gene as a selectable markerfor transforming Chorella ellipsoidea. Huang et al. (2007), Appl.Microbiol. Biotechnol. 72:197-205 (incorporated by reference herein inits entirety), reported on the use of Sh ble as a selectable marker forChlorella sp. DT.

V. Lipid Pathway Engineering

In addition to altering the ability of microorganisms (e.g., microalgae,oleaginous yeast, fungi, or bacteria), such as Prototheca to utilizefeedstocks such as sucrose-containing feedstocks, the present inventionalso provides recombinant microorganisms (e.g., Prototheca) that havebeen modified to alter the properties and/or proportions of lipidsproduced. The pathway can further, or alternatively, be modified toalter the properties and/or proportions of various lipid moleculesproduced through enzymatic processing of lipids and intermediates in thefatty acid pathway. In various embodiments, the recombinantmicroorganisms (e.g., Prototheca cells) of the invention have, relativeto their untransformed counterparts, optimized lipid yield per unitvolume and/or per unit time, carbon chain length (e.g., for renewablediesel production or for industrial chemicals applications requiringlipid feedstock), reduced number of double or triple bonds, optionallyto zero, and increasing the hydrogen:carbon ratio of a particularspecies of lipid or of a population of distinct lipid. In addition,microorganisms that produce desirable hydrocarbons can be engineered toproduce such components in higher quantities, or with greaterspecificity.

In the case of microalgae, some wild-type cells already have good growthcharacteristics but do not produce the desired types or quantities oflipids. Examples include, without limitation, Pyrobotrys, Phormidium,Agmenellum, Carteria, Lepocinclis, Pyrobotrys, Nitzschia, Lepocinclis,Anabaena, Euglena, Spirogyra, Chlorococcum, Tetraedron, Oscillatoria,Phagus, and Chlorogonium, which have the desirable growth characteristicof growing in municipal sewage or wastewater. Such cells, as well asspecies of Chlorella, Prototheca and other microbes, can be engineeredto have improved lipid production characteristics. Desiredcharacteristics include optimizing lipid yield per unit volume and/orper unit time, carbon chain length (e.g., for biodiesel production orfor industrial applications requiring hydrocarbon feedstock), reducingthe number of double or triple bonds, optionally to zero, removing oreliminating rings and cyclic structures, and increasing thehydrogen:carbon ratio of a particular species of lipid or of apopulation of distinct lipid. In addition, microalgae that produceappropriate hydrocarbons can also be engineered to have even moredesirable hydrocarbon outputs. Examples of such microalgae includespecies of the genus Chlorella and the genus Prototheca.

In particular embodiments, one or more key enzymes that control branchpoints in metabolism to fatty acid synthesis have been up-regulated ordown-regulated to improve lipid production. Up-regulation can beachieved, for example, by transforming cells with expression constructsin which a gene encoding the enzyme of interest is expressed, e.g.,using a strong promoter and/or enhancer elements that increasetranscription. Such constructs can include a selectable marker such thatthe transformants can be subjected to selection, which can result inamplification of the construct and an increase in the expression levelof the encoded enzyme. Examples of enzymes suitable for up-regulationaccording to the methods of the invention include pyruvatedehydrogenase, which plays a role in converting pyruvate to acetyl-CoA(examples, some from microalgae, include Genbank accession numbersNP_(—)415392; AAA53047; Q1XDM1; and CAF05587). Up-regulation of pyruvatedehydrogenase can increase production of acetyl-CoA, and therebyincrease fatty acid synthesis. Acetyl-CoA carboxylase catalyzes theinitial step in fatty acid synthesis. Accordingly, this enzyme can beup-regulated to increase production of fatty acids (examples, some frommicroalgae, include Genbank accession numbers BAA94752; AAA75528;AAA81471; YP_(—)537052; YP_(—)536879; NP_(—)045833; and BAA57908). Fattyacid production can also be increased by up-regulation of acyl carrierprotein (ACP), which carries the growing acyl chains during fatty acidsynthesis (examples, some from microalgae, include Genbank accessionnumbers AOTOF8; P51280; NP_(—)849041; YP_(—)874433).Glycerol-3-phosphate acyltransferase catalyzes the rate-limiting step offatty acid synthesis. Up-regulation of this enzyme can increase fattyacid production (examples, some from microalgae, include Genbankaccession numbers AAA74319; AAA33122; AAA37647; P44857; and ABO94442).

Up- and/or down-regulation of genes can be applied to global regulatorscontrolling the expression of the genes of the fatty acid biosyntheticpathways. Accordingly, one or more global regulators of fatty acidsynthesis can be up- or down-regulated, as appropriate, to inhibit orenhance, respectively, the expression of a plurality of fatty acidsynthetic genes and, ultimately, to increase lipid production. Examplesinclude sterol regulatory element binding proteins (SREBPs), such asSREBP-1a and SREBP-1c (for examples see Genbank accession numbersNP_(—)035610 and Q9WTN3).

The present invention also provides recombinant microorganisms (e.g.,Prototheca cells) that have been modified to contain one or moreexogenous genes encoding lipid modification enzymes such as, forexample, fatty acyl-ACP thioesterases (e.g., C. callophylla (SEQ ID NO:145 and SEQ ID NO: 146; see also Table 4), fatty acyl-CoA/aldehydereductases (see Table 6), fatty acyl-CoA reductases (see Table 7), fattyaldehyde decarbonylase (see Table 8), fatty aldehyde reductases,desaturases (such as stearoyl-ACP desaturases (e.g., a codon optimizedR. communis SAD, SEQ ID NO: 147 and SEQ ID NO: 148) and fatty acyldesaturases and squalene synthases (see GenBank Accession numberAF205791). In some embodiments, genes encoding a fatty acyl-ACPthioesterase and a naturally co-expressed acyl carrier protein aretransformed into a Prototheca cell, optionally with one or more genesencoding other lipid modification enzymes. In other embodiments, the ACPand the fatty acyl-ACP thioesterase may have an affinity for one anotherthat imparts an advantage when the two are used together in the microbesand methods of the present invention, irrespective of whether they areor are not naturally co-expressed in a particular tissue or organism.Thus, the present invention contemplates both naturally co-expressedpairs of these enzymes as well as those that share an affinity forinteracting with one another to facilitate cleavage of a length-specificcarbon chain from the ACP.

In still other embodiments, an exogenous gene encoding a desaturase istransformed into the microorganism (e.g., a Prototheca cell) inconjunction with one or more genes encoding other lipid modificationenzymes to provide modifications with respect to lipid saturation. Inother embodiments, an endogenous desaturase gene is overexpressed (e.g.,through the introduction of additional copies off the gene) in themicroorganism (e.g., a Prototheca cell). Stearoyl-ACP desaturase (see,e.g., GenBank Accession numbers AAF15308; ABM45911; and AAY86086), forexample, catalyzes the conversion of stearoyl-ACP to oleoyl-ACP.Up-regulation of this gene can increase the proportion ofmonounsaturated fatty acids produced by a cell; whereas down-regulationcan reduce the proportion of monounsaturates. For illustrative purposes,stearoyl-ACP desaturases (SAD) are responsible for the synthesis ofC18:1 fatty acids from C18:0 precursors. Another family of desaturasesare the fatty acyl desaturases (FAD), including delta 12 fatty aciddesaturases (Δ12 FAD). These desaturases also provide modifications withrespect to lipid saturation. For illustrative purposes, delta 12 fattyacid desaturases are responsible for the synthesis of C18:2 fatty acidsfrom C18:1 precursors. Similarly, the expression of one or moreglycerolipid desaturases can be controlled to alter the ratio ofunsaturated to saturated fatty acids such as ω-6 fatty acid desaturase,ω-3 fatty acid desaturase, or ω-6-oleate desaturase. In someembodiments, the desaturase can be selected with reference to a desiredcarbon chain length, such that the desaturase is capable of makinglocation specific modifications within a specified carbon-lengthsubstrate, or substrates having a carbon-length within a specifiedrange. In another embodiment, if the desired fatty acid profile is anincrease in monounsaturates (such as C16:1 and/or C18:1) overexpressionof a SAD or expression of a heterologous SAD can be coupled with thesilencing or inactivation (e.g., through mutation, RNAi, knockout of anendogenous desaturase gene, etc.) of a fatty acyl desaturase (FAD).

In other embodiments, the microorganism (e.g., Prototheca cell) has beenmodified to have a mutated endogenous desaturase gene, wherein themutation renders the gene or desaturase enzyme inactive. In some cases,the mutated endogenous desaturase gene is a fatty acid desaturase (FAD).In other cases, the mutated endogenous desaturase gene is a stearoylAcyl carrier protein desaturase (SAD). Example 11 below describes thetargeted ablation or knockout of stearoyl-ACP desaturases and delta 12fatty acid desaturases.

In some cases, it may be advantageous to pair one or more of the geneticengineering techniques in order to achieve a trangenic cell thatproduces the desired lipid profile. In one embodiment, a microorganism(e.g., a Prototheca cell) comprises a mutated endogenous desaturase geneand one or more exogenous gene. In non-limiting examples, a Protothecacell with a mutated endogenous desaturase gene can also express anexogenous fatty acyl-ACP thioesterase gene and/or a sucrose invertasegene. Example 11 below describes a transgenic Prototheca cell containinga targeted ablation or knockout of an endogenous SAD and also expressesa Cinnamomum camphora C14-preferring thioesterase and a sucroseinvertase. In this case, the transgenic Prototheca cell produces a lipidprofile that closely approximates the lipid profile found in tallow.Tallow is typically derived from rendered beef or mutton fat, is solidat room temperature and is utilized in a variety of applications in thefood, cosmetics, and chemicals industries. The fatty acid profile oftallow is: 4% C14:0; 26% C16:0; 3% C16:1; 14% C18:0; 41% C18:1; 3%C18:2; and 1% C18:3. As is shown in Example 11 below, clones oftransgenic Prototheca cells with a targeted ablation or knockout of anendogenous SAD and expressing a C. camphora C14-preferring thioesterasehave lipid profiles of: less than 1% C12 and shorter carbon chain lengthfatty acids; 2.74% to 6.13% C14:0; 23.07% to 25.69% C16:0; 7.02% to11.08% C18:0; 42.03% to 51.21% C18:1; and 9.37% to 13.45% C18:2(expressed in area percent). In some cases, the transgenic Protothecacells have lipid profiles of: 3-5% C14:0; 25-27% C16:0; 10-15% C18:0;and 40-45% C18:1.

Thus, in particular embodiments, microbes of the present invention aregenetically engineered to express one or more exogenous genes selectedfrom an acyl-ACP thioesterase, an acyl-CoA/aldehyde reductase, a fattyacyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehydedecarbonylase, or a naturally co-expressed acyl carrier protein.Suitable expression methods are described above with respect to theexpression of a lipase gene, including, among other methods, inducibleexpression and compartmentalized expression. A fatty acyl-ACPthioesterase cleaves a fatty acid from an acyl carrier protein (ACP)during lipid synthesis. Through further enzymatic processing, thecleaved fatty acid is then combined with a coenzyme to yield an acyl-CoAmolecule. This acyl-CoA is the substrate for the enzymatic activity of afatty acyl-CoA reductase to yield an aldehyde, as well as for a fattyacyl-CoA/aldehyde reductase to yield an alcohol. The aldehyde producedby the action of the fatty acyl-CoA reductase identified above is thesubstrate for further enzymatic activity by either a fatty aldehydereductase to yield an alcohol, or a fatty aldehyde decarbonylase toyield an alkane or alkene.

In some embodiments, fatty acids, glycerolipids, or the correspondingprimary alcohols, aldehydes, alkanes or alkenes, generated by themethods described herein, contain 8, 10, 12, or 14 carbon atoms.Preferred fatty acids for the production of diesel, biodiesel, renewablediesel, or jet fuel, or the corresponding primary alcohols, aldehydes,alkanes and alkenes, for industrial applications contain 8 to 14 carbonatoms. In certain embodiments, the above fatty acids, as well as theother corresponding hydrocarbon molecules, are saturated (with nocarbon-carbon double or triple bonds); mono unsaturated (single doublebond); poly unsturated (two or more double bonds); are linear (notcyclic) or branched. For fuel production, greater saturation ispreferred.

The enzymes described directly above have a preferential specificity forhydrolysis of a substrate containing a specific number of carbon atoms.For example, a fatty acyl-ACP thioesterase may have a preference forcleaving a fatty acid having 12 carbon atoms from the ACP. In someembodiments, the ACP and the length-specific thioesterase may have anaffinity for one another that makes them particularly useful as acombination (e.g., the exogenous ACP and thioesterase genes may benaturally co-expressed in a particular tissue or organism from whichthey are derived). Therefore, in various embodiments, the recombinantPrototheca cell of the invention can contain an exogenous gene thatencodes a protein with specificity for catalyzing an enzymatic activity(e.g., cleavage of a fatty acid from an ACP, reduction of an acyl-CoA toan aldehyde or an alcohol, or conversion of an aldehyde to an alkane)with regard to the number of carbon atoms contained in the substrate.The enzymatic specificity can, in various embodiments, be for asubstrate having from 8 to 34 carbon atoms, preferably from 8 to 18carbon atoms, and more preferably from 8 to 14 carbon atoms. A preferredspecificity is for a substrate having fewer, i.e., 12, rather than more,i.e., 18, carbon atoms.

Other fatty acyl-ACP thioesterases suitable for use with the microbesand methods of the invention include, without limitation, those listedin Table 4.

TABLE 4 Fatty acyl-ACP thioesterases and GenBank accession numbers.Umbellularia californica fatty acyl-ACP thioesterase (GenBank #AAC49001)(SEQ ID NO: 203) Cinnamomum camphora fatty acyl-ACP thioesterase(GenBank #Q39473) Umbellularia californica fatty acyl-ACP thioesterase(GenBank #Q41635) Myristica fragrans fatty acyl-ACP thioesterase(GenBank #AAB71729) (SEQ ID NO: 224) Myristica fragrans fatty acyl-ACPthioesterase (GenBank #AAB71730) (SEQ ID NO: 222) Elaeis guineensisfatty acyl-ACP thioesterase (GenBank #ABD83939) (SEQ ID NO: 204) Elaeisguineensis fatty acyl-ACP thioesterase (GenBank #AAD42220) Populustomentosa fatty acyl-ACP thioesterase (GenBank #ABC47311) (SEQ ID NO:207) Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank#NP_172327) (SEQ ID NO: 208) Arabidopsis thaliana fatty acyl-ACPthioesterase (GenBank #CAA85387) (SEQ ID NO: 209) Arabidopsis thalianafatty acyl-ACP thioesterase (GenBank #CAA85388) (SEQ ID NO: 210)Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank #Q9SQI3) (SEQ IDNO: 211) Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank#CAA54060) (SEQ ID NO: 212) Cuphea hookeriana fatty acyl-ACPthioesterase (GenBank #AAC72882) (SEQ ID NO: 202) Cuphea calophyllasubsp. mesostemon fatty acyl-ACP thioesterase (GenBank #ABB71581) (SEQID NO: 213) Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank#CAC19933) Elaeis guineensis fatty acyl-ACP thioesterase (GenBank#AAL15645) (SEQ ID NO: 206) Cuphea hookeriana fatty acyl-ACPthioesterase (GenBank #Q39513) Gossypium hirsutum fatty acyl-ACPthioesterase (GenBank #AAD01982) (SEQ ID NO: 214) Vitis vinifera fattyacyl-ACP thioesterase (GenBank #CAN81819) (SEQ ID NO: 215) Garciniamangostana fatty acyl-ACP thioesterase (GenBank #AAB51525) Brassicajuncea fatty acyl-ACP thioesterase (GenBank #ABI18986) (SEQ ID NO: 216)Madhuca longifolia fatty acyl-ACP thioesterase (GenBank #AAX51637) (SEQID NO: 217) Brassica napus fatty acyl-ACP thioesterase (GenBank#ABH11710) Oryza sativa (indica cultivar-group) fatty acyl-ACPthioesterase (GenBank #EAY86877) (SEQ ID NO: 218) Oryza sativa (japonicacultivar-group) fatty acyl-ACP thioesterase (GenBank #NP_001068400) (SEQID NO: 219) Oryza sativa (indica cultivar-group) fatty acyl-ACPthioesterase (GenBank #EAY99617) (SEQ ID NO: 220) Cuphea hookerianafatty acyl-ACP thioesterase (GenBank #AAC49269) Ulmus Americana fattyacyl-ACP thioesterase (GenBank #AAB71731) Cuphea lanceolata fattyacyl-ACP thioesterase (GenBank #CAB60830) (SEQ ID NO: 221) Cupheapalustris fatty acyl-ACP thioesterase (GenBank #AAC49180) Iris germanicafatty acyl-ACP thioesterase (GenBank #AAG43858) Iris germanica fattyacyl-ACP thioesterase (GenBank #AAG43858.1) Cuphea palustris fattyacyl-ACP thioesterase (GenBank #AAC49179) Myristica fragrans fattyacyl-ACP thioesterase (GenBank# AAB71729) Myristica fragrans fattyacyl-ACP thioesterase (GenBank# AAB717291.1) Cuphea hookeriana fattyacyl-ACP thioesterase (GenBank #U39834) (SEQ ID NO: 197) Umbelluariacalifornica fatty acyl-ACP thioesterase (GenBank # M94159) (SEQ ID NO:285) Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank #U31813)(SEQ ID NO: 223) Cuphea wrightii fatty acyl-ACOP thioesterase (GenBank#U56103) (SEQ ID NO: 183) Ricinus communis fatty acyl-ACP thioesterase(GenBank #ABS30422) (SEQ ID NO: 198)

The Examples below describe the successful targeting and expression ofheterologous fatty acyl-ACP thioesterases from Cuphea hookeriana,Umbellularia californica, Cinnamomun camphora, Cuphea palustris, Cuphealanceolata, Iris germanica, Myristica fragrans and Ulmus americana inPrototheca species. Additionally, alterations in fatty acid profileswere confirmed in the host cells expression these heterologous fattyacyl-ACP thioesterases. These results were quite unexpected given thelack of sequence identity between algal and higher plant thioesterasesin general, and between Prototheca moriformis fatty acyl-ACPthioesterase and the above listed heterologous fatty acyl-ACPthioesterases. As shown in the Examples, the expression of theseheterologous thioesterases in Prototheca generates a transgenicmicroalgae that is able to produce oil/lipids with truly unique fattyacid profiles that are currently not available from commercial seedcrops, even through the blending of several seed crop oils. Table 5shows the fatty acid profiles of common commercial seed oils. Allcommercial seed oil data below were compiled from the US PharmacopeiasFood and Chemicals Codes, 7^(th) Ed. 2010-2011. Tallow data is from theNational Research Council: Fat Content and Composition of AnimalProducts (1976).

TABLE 5 Lipid profiles of commercial seed oils (in percentages). C18:0-C18:1- C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 diOH OH C18:2 C18:3 α R.communis 0 0 0 0  0.9-1.6 1.0-1.8 3.7-6.7 0.4-1.3 83.6-89.0 0 0.2-0.6(Castor oil) C. nucifera 5.0-9.0 4.0-8.0 44-52 15-21  8.0-11.0  1.0-4.0 5.0-8.0 0 0  0-2.5 0 (Coconut oil) Z. mays 0 0 0 <1.0  8.0-19.0 0.5-4.0  19-50 0 0 38-65 <2.0 (Corn oil) G. 0 0 <0.1 0.5-2.0 17-29 1.0-4.0  13-44 0 0 40-63 0.1-2.1  barbadense (Cottonseed oil) B. rapa,B 0 0 <0.1 <0.2 <6.0 <2.5 >50 0 0 <40 <14 napus, B. juncea (Canola) O.europea 0 0 0 <0.1  6.5-20.0  0.5-5.0  56-85 0 0  3.5-20.0 <1.2 (Olive)A. hypogaea 0 0 <0.1 <0.2  7.0-16.0  1.3-6.5  35-72 0 0 13.0-43  <0.6(Peanut) E. guineensis 3.0-5.0 2.5-6.0 40-52 14.0-18.0  7.0-10.0 1.0-3.0 11.0-19.0 0 0 0.5-4.0  0 (Palm kernel) E. guineensis 0 0 00.5-5.9 32.0-47.0  2.0-8.0  34-44 0 0 7.2-12.0 0 (Palm) C. tinctorus 0 0<0.1 <0.1  2.0-10.0  1.0-10.0  7.0-16.0 0 0 72-81  <1.5 (Safflower) H.annus 0 0 <0.1 <0.5  3.0-10.0  1.0-10.0  14-65 0 0 20-75  <0.5(Sunflower) G. max 0 0 <0.1 <0.5  7.0-12.0  2.0-5.5  19-30 0 0 48-65 5.0-10.0 (Soybean) L. 0 0 <0.1 <0.5  2.0-9.0  2.0-5.0 8.0-60 0 0 40-80 <5.0 usitatissimum (Solin-Flax) B. parkii 0 0 0 0  3.8-4.1 41.2-56.834.0-46.9 0 0 3.7-6.5  0 (Sheanut) Cocoa Butter 0-1 0-1 0-4  22-30 24-3729-38 0-3  Tallow 3-4  23-28 14-23 36-43 1-4  <1 Lard 1-2  22-26 13-1839-45 8-15 0.5-1.5 

As an example, none of these common seed oils contain high amounts of C8or C10 fatty acids, with coconut oil and palm kernel oil being thelargest sources, but both a ratio of 1:1 (C8:C10 fatty acids). As shownin the Examples, Prototheca transformed with Cuphea palustris C:8preferring thioesterase was able to achieve not only a C8 fatty acidlevels of over 12%, but also, the ratio of C8:C10 fatty acids were abouta 5:1. Changes in fatty acid levels are useful for producing oilscontaining a tailored fatty acid profile for a variety of commercialapplications. Additionally, changes of ratios between different fattyacid chain lengths is something has not been available commercially inoils that have not been through further costly chemical processes (suchas esterification, distillation, fractionation, and re-esterification).As another example, palm oil is the highest C16:0 fatty acid (32-47%)containing oils, but palm oil has very little C14:0 fatty acids.Prototheca containing the U. americana thioesterase achieved about33-38% C16:0 fatty acids and about a 10-16% C14:0 fatty acids (about a2:1 C16:0 to C14:0 ratio). This fatty acid profile is unachievablethrough blending of existing oils at a commercial level because the seedoils that are high in 16:0 fatty acids usually do not contain much 14:0fatty acids.

The Examples below also describe, for the first time, the successfultargeting and expression of at least two fatty acyl-ACP thioesterases inone clone. The alterations in the fatty acid profiles were confirmed inthese clones and depending on which two thioesterases were co-expressedin one clone, the fatty acid profiles were impacted in different ways.As an example, from Table 5 above, both coconut oil and palm kernel oilhave C12:C14 ratios of roughly 3:1. As described in the Examples below,a Prototheca transformant containing two heterologous thioesterase geneswas able to produce C12:C14 fatty acid levels at a ratio of roughly 5:1.This kind of ratio of C12:C14 fatty acids has been, up to now,unachievable at commercial levels (i.e., through blending of seed oils).

Another novel aspect of the oils produced by transgenic microalgae isthe degree of saturation of the fatty acids. Palm oil is currently thelargest source of saturated oil, with a total saturates to unsaturatesof 52% to 48%. As shown in the Examples below, Prototheca withheterologous thioesterases from U. americana and C. camphora achievedtotal saturates levels of over 60% in the oil that it produced. Alsoshown in the Examples below, Prototheca with heterologous thioesterasefrom U. americana achieved total saturates level of over 86% in the oilthat it produced.

Fatty acyl-CoA/aldehyde reductases suitable for use with the microbesand methods of the invention include, without limitation, those listedin Table 6.

TABLE 6 Fatty acyl-CoA/aldehyde reductases listed by GenBank accessionnumbers. AAC45217, YP_047869, BAB85476, YP_001086217, YP_580344,YP_001280274, YP_264583, YP_436109, YP_959769, ZP_01736962, ZP_01900335,ZP_01892096, ZP_01103974, ZP_01915077, YP_924106, YP_130411,ZP_01222731, YP_550815, YP_983712, YP_001019688, YP_524762, YP_856798,ZP_01115500, YP_001141848, NP_336047, NP_216059, YP_882409, YP_706156,YP_001136150, YP_952365, ZP_01221833, YP_130076, NP_567936, AAR88762,ABK28586, NP_197634, CAD30694, NP_001063962, BAD46254, NP_001030809,EAZ10132, EAZ43639, EAZ07989, NP_001062488, CAB88537, NP_001052541,CAH66597, CAE02214, CAH66590, CAB88538, EAZ39844, AAZ06658, CAA68190,CAA52019, and BAC84377

Fatty acyl-CoA reductases suitable for use with the microbes and methodsof the invention include, without limitation, those listed in Table 7.

TABLE 7 Fatty acyl-CoA reductases listed by GenBank accession numbers.NP_187805, ABO14927, NP_001049083, CAN83375, NP_191229, EAZ42242,EAZ06453, CAD30696, BAD31814, NP_190040, AAD38039, CAD30692, CAN81280,NP_197642, NP_190041, AAL15288, and NP_190042

Fatty aldehyde decarbonylases suitable for use with the microbes andmethods of the invention include, without limitation, those listed inTable 8.

TABLE 8 Fatty aldehyde decarbonylases listed by GenBank accessionnumbers. NP_850932, ABN07985, CAN60676, AAC23640, CAA65199, AAC24373,CAE03390, ABD28319, NP_181306, EAZ31322, CAN63491, EAY94825, EAY86731,CAL55686, XP_001420263, EAZ23849, NP_200588, NP_001063227, CAN83072,AAR90847, and AAR97643

Combinations of naturally co-expressed fatty acyl-ACP thioesterases andacyl carrier proteins are suitable for use with the microbes and methodsof the invention.

Additional examples of hydrocarbon or lipid modification enzymes includeamino acid sequences contained in, referenced in, or encoded by nucleicacid sequences contained or referenced in, any of the following U.S.Pat. Nos. 6,610,527; 6,451,576; 6,429,014; 6,342,380; 6,265,639;6,194,185; 6,114,160; 6,083,731; 6,043,072; 5,994,114; 5,891,697;5,871,988; 6,265,639, and further described in GenBank Accessionnumbers: AA018435; ZP_(—)00513891; Q38710; AAK60613; AAK60610; AAK60611;NP_(—)113747; CAB75874; AAK60612; AAF20201; BAA11024; AF205791; andCAA03710.

Other enzymes in the lipid biosynthetic pathways are also suitable foruse with microbes and methods of the invention. For example, ketoacyl-ACP synthase (Kas) enzymes work in conjunction with some of theabove listed enzymes in the lipid biosynthetic pathway. There differentclasses of Kas enzymes: Kas I participates in successive condensationsteps between the ever-growing acyl ACP chains and malonyl-ACP. Kas IItypically participates in the final condensation step leading fromC16:0-ACP to C18:0-ACP incorporating malonyl-ACP. As such, in higherplants and some microalgae species/strains that synthesize predominantlyC16-C18:0 fatty acids (and their unsaturated derivatives), Kas IIenzymes interact with products of FatA genes (acyl-ACP thioesterases).

Acyl-ACP thioesterases are the terminators of higher plant (and somemicroalgal species) fatty acid biosynthesis, and in most plant species,this is carried out by members of the FatA gene family, whose role is toterminate elongation at the C16:0 to C18:0 stage. In species thatsynthesize shorter chain fatty acids (such as Cuphea, Elaeis, Myristica,or Umbellularia), a different group of acyl-ACP thioesterases encoded byFatB genes carry out this termination step (see e.g., the codonoptimized coding region of Cocos nucifera FatB3-B, SEQ ID NO: 189). Theinteraction between Kas II enzymes and acyl-Acp thioesterases isimportant for the correct termination of fatty acid chain elongation. Asa consequence, in higher plant species (and microalgal species) thathave evolved FatB genes capable of shorter chain lipid biosynthesis,there has been a corresponding co-evolution of an additional class ofKas genes, termed Kas IV genes. Kas IV genes are responsible for chainlength elongation of a specific size range of fatty acids, 4-14 carbonsin length.

Other suitable enzymes for use with the microbes and the methods of theinvention include those that have at least 70% amino acid identity withone of the proteins listed in Tables 4, 6-8, and that exhibit thecorresponding desired enzymatic activity (e.g., cleavage of a fatty acidfrom an acyl carrier protein, reduction of an acyl-CoA to an aldehyde oran alcohol, or conversion of an aldehyde to an alkane). In additionalembodiments, the enzymatic activity is present in a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% identity with one of theabove described sequences, all of which are hereby incorporated byreference as if fully set forth.

By selecting the desired combination of exogenous genes to be expressed,one can tailor the product generated by the microbe, which may then beextracted from the aqueous biomass. For example, the microbe cancontain: (i) an exogenous gene encoding a fatty acyl-ACP thioesterase;and, optionally, (ii) a naturally co-expressed acyl carrier protein oran acyl carrier protein otherwise having affinity for the fatty acyl-ACPthioesterase (or conversely); and, optionally, (iii) an exogenous geneencoding a fatty acyl-CoA/aldehyde reductase or a fatty acyl-CoAreductase; and, optionally, (iv) an exogenous gene encoding a fattyaldehyde reductase or a fatty aldehyde decarbonylase. The microbe, underculture conditions described herein, synthesizes a fatty acid linked toan ACP and the fatty acyl-ACP thioesterase catalyzes the cleavage of thefatty acid from the ACP to yield, through further enzymatic processing,a fatty acyl-CoA molecule. When present, the fatty acyl-CoA/aldehydereducatase catalyzes the reduction of the acyl-CoA to an alcohol.Similarly, the fatty acyl-CoA reductase, when present, catalyzes thereduction of the acyl-CoA to an aldehyde. In those embodiments in whichan exogenous gene encoding a fatty acyl-CoA reductase is present andexpressed to yield an aldehyde product, a fatty aldehyde reductase,encoded by the third exogenous gene, catalyzes the reduction of thealdehyde to an alcohol Similarly, a fatty aldehyde decarbonylasecatalyzes the conversion of the aldehyde to an alkane or an alkene, whenpresent.

In another embodiment, the microbe can contain: (i) an exogenous geneencoding a fatty acyl-ACP thioesterase; (ii) optionally, a naturallyco-expressed acyl carrier protein or an acyl carrier protein havingaffinity for the fatty acid acyl-ACP thioesterase; (iii) a mutatedendogenous desaturase gene, wherein the mutation renders the desaturasegene or desaturase protein inactive, such as a desaturase knockout; (iv)overexpression of an endogenous stearoyl acyl carrier protein desaturaseor the expression of a heterologous SAD; and (v) any combination of theforegoing.

Genes encoding such enzymes, such as fatty acyl ACP thioesterases, canbe obtained from cells already known to exhibit significant lipidproduction such as Chlorella protothecoides. Genes already known to havea role in lipid production, e.g., a gene encoding an enzyme thatsaturates double bonds, can be transformed individually into recipientcells. However, to practice the invention it is not necessary to make apriori assumptions as to which genes are required. Methods foridentifiying genes that can alter (improve) lipid production inmicroalgae are described in PCT Pub. No. 2008/151149.

Thus, the present invention provides a microorganism (e.g., a Protothecacell) that has been genetically engineered to express a lipid pathwayenzyme at an altered level compared to a wild-type cell of the samespecies. In some cases, the cell produces more lipid compared to thewild-type cell when both cells are grown under the same conditions. Insome cases, the cell has been genetically engineered and/or selected toexpress a lipid pathway enzyme at a higher level than the wild-typecell. In some cases, the lipid pathway enzyme is selected from the groupconsisting of pyruvate dehydrogenase, acetyl-CoA carboxylase, acylcarrier protein, and glycerol-3 phosphate acyltransferase. In somecases, the cell has been genetically engineered and/or selected toexpress a lipid pathway enzyme at a lower level than the wild-type cell.In at least one embodiment in which the cell expresses the lipid pathwayenzyme at a lower level, the lipid pathway enzyme comprises citratesynthase.

In some embodiments, the cell has been genetically engineered and/orselected to express a global regulator of fatty acid synthesis at analtered level compared to the wild-type cell, whereby the expressionlevels of a plurality of fatty acid synthetic genes are altered comparedto the wild-type cell. In some cases, the lipid pathway enzyme comprisesan enzyme that modifies a fatty acid. In some cases, the lipid pathwayenzyme is selected from a stearoyl-ACP desaturase and a glycerolipiddesaturase. In some cases, the cell has been genetically engineeredand/or selected to express a lower level of a lipid pathway enzyme, ornot to express a specific lipid pathway enzyme at all (i.e., wherein alipid pathway enzyme has been knockout, or replaced with an exogenousgene).

Some microalgae produce significant quantities of non-lipid metabolites,such as, for example, polysaccharides. Because polysaccharidebiosynthesis can use a significant proportion of the total metabolicenergy available to cells, mutagenesis of lipid-producing cells followedby screening for reduced or eliminated polysaccharide productiongenerates novel strains that are capable of producing higher yields oflipids.

In other embodiments, the present invention is directed to anoil-producing microbe containing one or more exogenous genes, whereinthe exogenous genes encode protein(s) selected from the group consistingof a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, a fattyaldehyde reductase, a fatty acyl-CoA/aldehyde reductase, a fattyaldehyde decarbonylase, a desaturase, and an acyl carrier protein. Inanother embodiment, an endogenous desaturase gene is overexpressed in amicro containing one or more of the above exogenous genes. In oneembodiment, the exogenous gene is in operable linkage with a promoter,which is inducible or repressible in response to a stimulus. In somecases, the stimulus is selected from the group consisting of anexogenously provided small molecule, heat, cold, and limited or nonitrogen in the culture media. In some cases, the exogenous gene isexpressed in a cellular compartment. In some embodiments, the cellularcompartment is selected from the group consisting of a chloroplast, aplastid and a mitochondrion. In some embodiments the microbe isPrototheca moriformis, Prototheca krugani, Prototheca stagnora orPrototheca zopfii.

In one embodiment, the exogenous gene encodes a fatty acid acyl-ACPthioesterase. In some cases, the thioesterase encoded by the exogenousgene catalyzes the cleavage of an 8 to 18-carbon fatty acid from an acylcarrier protein (ACP). In some cases, the thioesterase encoded by theexogenous gene catalyzes the cleavage of a 10 to 14-carbon fatty acidfrom an ACP. In one embodiment, the thioesterase encoded by theexogenous gene catalyzes the cleavage of a 12-carbon fatty acid from anACP.

In one embodiment, the exogenous gene encodes a fatty acyl-CoA/aldehydereductase. In some cases, the reductase encoded by the exogenous genecatalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to acorresponding primary alcohol. In some cases, the reductase encoded bythe exogenous gene catalyzes the reduction of a 10 to 14-carbon fattyacyl-CoA to a corresponding primary alcohol. In one embodiment, thereductase encoded by the exogenous gene catalyzes the reduction of a12-carbon fatty acyl-CoA to dodecanol.

The present invention also provides a recombinant Prototheca cellcontaining two exogenous genes, wherein a first exogenous gene encodes afatty acyl-ACP thioesterase and a second exogenous gene encodes aprotein selected from the group consisting of a fatty acyl-CoAreductase, a fatty acyl-CoA/aldehyde reductase, and an acyl carrierprotein. In some cases, the two exogenous genes are each in operablelinkage with a promoter, which is inducible in response to a stimulus.In some cases, each promoter is inducible in response to an identicalstimulus, such as limited or no nitrogen in the culture media.Limitation or complete lack of nitrogen in the culture media stimulatesoil production in some microorganisms such as Prototheca species, andcan be used as a trigger to inducec oil production to high levels. Whenused in combination with the genetic engineering methods disclosedherein, the lipid as a percentage of dry cell weight can be pushed tohigh levels such as at least 30%, at least 40%, at least 50%, at least60%, at least 70% and at least 75%; methods disclosed herein provide forcells with these levels of lipid, wherein the lipid is at least 1%-5%,preferably at least 4%, C8-C14, at least 0.25%-1%, preferably at least0.3%, C8, at least 1%-5%, preferably at least 2%, C10, at least 1%-5%,preferably at least 2%, C12, and at least 1%-5%, preferably at least 2%,C14. In some embodiments the cells are over 10%, over 15%, over 20%, orover 25% lipid by dry cell weight and contain lipid that is at least 5%,at least 10% or at least 15% C8-C14, at least 10%, at least 15%, atleast 20%, at least 25% or at least 30% C8-C14, at least 20%, at least25%, at least 30%, at least 35% or at least 40%, C8-C14, 5%-40%,preferably 10-30%, C8-C14 and 10%-40%, preferably 20-30%, C8-C14.

The novel oils disclosed herein are distinct from other naturallyoccurring oils that are high in mid-chain fatty acids, such as palm oil,palm kernel oil, and coconut oil. For example, levels of contaminantssuch as carotenoids are far higher in palm oil and palm kernel oil thanin the oils of the invention. Palm and palm kernel oils in particularcontain alpha and beta carotenes and lycopene in much higher amountsthan is in the oils of the invention. In addition, over 20 differentcarotenoids are found in palm and palm kernel oil, whereas the Examplesdemonstrate that the oils of the invention contain very few carotenoidsspecies and very low levels. In addition, the levels of vitamin Ecompounds such as tocotrienols are far higher in palm, palm kernel, andcoconut oil than in the oils of the invention.

In one embodiment, the thioesterase encoded by the first exogenous genecatalyzes the cleavage of an 8 to 18-carbon fatty acid from an ACP. Insome embodiments, the second exogenous gene encodes a fattyacyl-CoA/aldehyde reductase which catalyzes the reduction of an 8 to18-carbon fatty acyl-CoA to a corresponding primary alcohol. In somecases, the thioesterase encoded by the first exogenous gene catalyzesthe cleavage of a 10 to 14-carbon fatty acid from an ACP, and thereductase encoded by the second exogenous gene catalyzes the reductionof a 10 to 14-carbon fatty acyl-CoA to the corresponding primaryalcohol, wherein the thioesterase and the reductase act on the samecarbon chain length. In one embodiment, the thioesterase encoded by thefirst exogenous gene catalyzes the cleavage of a 12-carbon fatty acidfrom an ACP, and the reductase encoded by the second exogenous genecatalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol. Insome embodiments, the second exogenous gene encodes a fatty acyl-CoAreductase which catalyzes the reduction of an 8 to 18-carbon fattyacyl-CoA to a corresponding aldehyde. In some embodiments, the secondexogenous gene encodes an acyl carrier protein that is naturallyco-expressed with the fatty acyl-ACP thioesterase.

In some embodiments, the second exogenous gene encodes a fatty acyl-CoAreductase, and the microbe further contains a third exogenous geneencoding a fatty aldehyde decarbonylase. In some cases, the thioesteraseencoded by the first exogenous gene catalyzes the cleavage of an 8 to18-carbon fatty acid from an ACP, the reductase encoded by the secondexogenous gene catalyzes the reduction of an 8 to 18-carbon fattyacyl-CoA to a corresponding fatty aldehyde, and the decarbonylaseencoded by the third exogenous gene catalyzes the conversion of an 8 to18-carbon fatty aldehyde to a corresponding alkane, wherein thethioesterase, the reductase, and the decarbonylase act on the samecarbon chain length.

In some embodiments, the second exogenous gene encodes an acyl carrierprotein, and the microbe further contains a third exogenous geneencoding a protein selected from the group consisting of a fattyacyl-CoA reductase and a fatty acyl-CoA/aldehyde reductase. In somecases, the third exogenous gene encodes a fatty acyl-CoA reductase, andthe microbe further contains a fourth exogenous gene encoding a fattyaldehyde decarbonylase.

The present invention also provides methods for producing an alcoholcomprising culturing a population of recombinant microorganisms (e.g.,Prototheca cells) in a culture medium, wherein the cells contain (i) afirst exogenous gene encoding a fatty acyl-ACP thioesterase, and (ii) asecond exogenous gene encoding a fatty acyl-CoA/aldehyde reductase, andthe cells synthesize a fatty acid linked to an acyl carrier protein(ACP), the fatty acyl-ACP thioesterase catalyzes the cleavage of thefatty acid from the ACP to yield, through further processing, a fattyacyl-CoA, and the fatty acyl-CoA/aldehyde reductase catalyzes thereduction of the acyl-CoA to an alcohol.

The present invention also provides methods of producing a lipidmolecule in a microorganism (e.g., a Prototheca cell). In oneembodiment, the method comprises culturing a population of Protothecacells in a culture medium, wherein the cells contain (i) a firstexogenous gene encoding a fatty acyl-ACP thioesterase, and (ii) a secondexogenous gene encoding a fatty acyl-CoA reductase, and wherein themicrobes synthesize a fatty acid linked to an acyl carrier protein(ACP), the fatty acyl-ACP thioesterase catalyzes the cleavage of thefatty acid from the ACP to yield, through further processing, a fattyacyl-CoA, and the fatty acyl-CoA reductase catalyzes the reduction ofthe acyl-CoA to an aldehyde.

The present invention also provides methods of producing a fatty acidmolecule having a specified carbon chain length in a microorganism(e.g., a Prototheca cell). In one embodiment, the method comprisesculturing a population of lipid-producing Prototheca cells in a culturemedium, wherein the microbes contain an exogenous gene encoding a fattyacyl-ACP thioesterase having an activity specific or preferential to acertain carbon chain length, such as 8, 10, 12 or 14 carbon atoms, andwherein the microbes synthesize a fatty acid linked to an acyl carrierprotein (ACP) and the thioesterase catalyzes the cleavage of the fattyacid from the ACP when the fatty acid has been synthesized to thespecific carbon chain length.

In the various embodiments described above, the microorganism (e.g., aPrototheca cell) can contain at least one exogenous gene encoding alipid pathway enzyme. In some cases, the lipid pathway enzyme isselected from the group consisting of a stearoyl-ACP desaturase, aglycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoAcarboxylase, an acyl carrier protein, and a glycerol-3 phosphateacyltransferase. In other cases, the microorganism (e.g., Protothecacell) contains a lipid modification enzyme selected from the groupconsisting of a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehydereductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, afatty aldehyde decarbonylase, and/or an acyl carrier protein.

A number of exemplary transformation cassettes or constructs used toexpress a variety of the lipid pathway enzymes and lipid modificationenzymes discussed herein are presented in the Examples. Other usefulconstructs, without limitation, are listed in Table 8A, below.

TABLE 8A Exemplary transformation constructs, codon-optimized codingregions, and enzymes. SEQ ID Transformation Construct/Codingregion/Enzyme NO C. hookeriana C10:0 specific thioesterase construct 243coding region for C. hookeriana C10:0 specific thioesterase 244(codon-optimized) C. hookeriana KAS IV enzyme construct 245 codingregion for C. hookeriana KAS IV enzyme (codon- 246 optimized) C.hookeriana KAS IV enzyme 247 C. hookeriana C10:0 specific thioesteraseplus C. hookeriana 248 KAS IV enzyme construct coding region for C.lanceolata C10:0 specific thioesterase with 249 UTEX 1435 Δ12 fatty aciddesaturase U. californica C12:0 specific thioesterase construct 250coding region for U. californica C12:0 specific thioesterase 251(codon-optimized) G. mangostana C16:0 thioesterase construct 252 codingregion for G. mangostana C16:0 thioesterase (codon- 253 optimized) B.napus C18:0 thioesterase construct 254 coding region for B. napus C18:0thioesterase (codon- 255 optimized) O. europaea stearoyl-ACP desaturaseconstruct 256 coding region for O. europaea stearoyl-ACP desaturase 257(codon-optimized) C. hookeriana C16:0 thioesterase construct 258 codingregion for C. hookeriana C16:0 thioesterase 259 (codon-optimized) E.guineensis C16:0 thioesterase construct 260 coding region for E.guineensis C16:0 thioesterase 261 (codon-optimized) C. tinctoriusACP-thioesterase at Δ12 fatty acid desaturase 262 locus construct codingregion for C. tinctorius ACP-thioesterase 263 (codon-optimized) M.fragrans C14:0-C18:0 broad specificity thioesterase 264 construct codingregion for M. fragrans C14:0-C18:0 broad specificity 265 thioesterase(codon-optimized) coding region for M. fragrans C:14:0 specificthioesterase 266 M. fragrans C14:0 specific thioesterase with Δ12 FAD267 transit peptide Ricinus communis ACP-thioesterase construct 268coding region for Ricinus communis ACP-thioesterase 269(codon-optimized) C. camphora C14:0 thioesterase construct 270 codingregion for C. camphora C14:0 thioesterase 271 (codon-optimized) C.camphora C14:0 specific thioesterase construct 272 C. camphora C14:0specific thioesterase construct 273 U. Americana C10:0-C16:0 specificthioesterase in a SAD locus 274 coding region for U. AmericanaC10:0-C16:0 specific 275 thioesterase (codon-optimized) C. wrightiiKASA1 + C. wrightii FatB2 thioesterase + suc2 276 construct codingregion for C. wrightii KASA1 (codon-optimized) 277 coding region for C.wrightii FatB2 thioesterase (codon- 278 optimized)

VI. Fuels and Chemicals Production

For the production of fuel in accordance with the methods of theinvention lipids produced by cells of the invention are harvested, orotherwise collected, by any convenient means. Lipids can be isolated bywhole cell extraction. The cells are first disrupted, and thenintracellular and cell membrane/cell wall-associated lipids as well asextracellular hydrocarbons can be separated from the cell mass, such asby use of centrifugation as described above. Intracellular lipidsproduced in microorganisms are, in some embodiments, extracted afterlysing the cells of the microorganism. Once extracted, the lipids arefurther refined to produce oils, fuels, or oleochemicals.

After completion of culturing, the microorganisms can be separated fromthe fermentation broth. Optionally, the separation is effected bycentrifugation to generate a concentrated paste. Centrifugation does notremove significant amounts of intracellular water from themicroorganisms and is not a drying step. The biomass can then optionallybe washed with a washing solution (e.g., DI water) to get rid of thefermentation broth and debris. Optionally, the washed microbial biomassmay also be dried (oven dried, lyophilized, etc.) prior to celldisruption. Alternatively, cells can be lysed without separation fromsome or all of the fermentation broth when the fermentation is complete.For example, the cells can be at a ratio of less than 1:1 v:v cells toextracellular liquid when the cells are lysed.

Microorganisms containing a lipid can be lysed to produce a lysate. Asdetailed herein, the step of lysing a microorganism (also referred to ascell lysis) can be achieved by any convenient means, includingheat-induced lysis, adding a base, adding an acid, using enzymes such asproteases and polysaccharide degradation enzymes such as amylases, usingultrasound, mechanical lysis, using osmotic shock, infection with alytic virus, and/or expression of one or more lytic genes. Lysis isperformed to release intracellular molecules which have been produced bythe microorganism. Each of these methods for lysing a microorganism canbe used as a single method or in combination simultaneously orsequentially. The extent of cell disruption can be observed bymicroscopic analysis. Using one or more of the methods described herein,typically more than 70% cell breakage is observed. Preferably, cellbreakage is more than 80%, more preferably more than 90% and mostpreferred about 100%.

In particular embodiments, the microorganism is lysed after growth, forexample to increase the exposure of cellular lipid and/or hydrocarbonfor extraction or further processing. The timing of lipase expression(e.g., via an inducible promoter) or cell lysis can be adjusted tooptimize the yield of lipids and/or hydrocarbons. Below are described anumber of lysis techniques. These techniques can be used individually orin combination.

In one embodiment of the present invention, the step of lysing amicroorganism comprises heating of a cellular suspension containing themicroorganism. In this embodiment, the fermentation broth containing themicroorganisms (or a suspension of microorganisms isolated from thefermentation broth) is heated until the microorganisms, i.e., the cellwalls and membranes of microorganisms degrade or breakdown. Typically,temperatures applied are at least 50° C. Higher temperatures, such as,at least 30° C. at least 60° C., at least 70° C., at least 80° C., atleast 90° C., at least 100° C., at least 110° C., at least 120° C., atleast 130° C. or higher are used for more efficient cell lysis. Lysingcells by heat treatment can be performed by boiling the microorganism.Alternatively, heat treatment (without boiling) can be performed in anautoclave. The heat treated lysate may be cooled for further treatment.Cell disruption can also be performed by steam treatment, i.e., throughaddition of pressurized steam. Steam treatment of microalgae for celldisruption is described, for example, in U.S. Pat. No. 6,750,048. Insome embodiments, steam treatment may be achieved by sparging steam intothe fermentor and maintaining the broth at a desired temperature forless than about 90 minutes, preferably less than about 60 minutes, andmore preferably less than about 30 minutes.

In another embodiment of the present invention, the step of lysing amicroorganism comprises adding a base to a cellular suspensioncontaining the microorganism. The base should be strong enough tohydrolyze at least a portion of the proteinaceous compounds of themicroorganisms used. Bases which are useful for solubilizing proteinsare known in the art of chemistry. Exemplary bases which are useful inthe methods of the present invention include, but are not limited to,hydroxides, carbonates and bicarbonates of lithium, sodium, potassium,calcium, and mixtures thereof. A preferred base is KOH. Base treatmentof microalgae for cell disruption is described, for example, in U.S.Pat. No. 6,750,048.

In another embodiment of the present invention, the step of lysing amicroorganism comprises adding an acid to a cellular suspensioncontaining the microorganism. Acid lysis can be effected using an acidat a concentration of 10-500 mN or preferably 40-160 nM. Acid lysis ispreferably performed at above room temperature (e.g., at 40-160°, andpreferably a temperature of 50-130°. For moderate temperatures (e.g.,room temperature to 100° C. and particularly room temperature to 65°,acid treatment can usefully be combined with sonication or other celldisruption methods.

In another embodiment of the present invention, the step of lysing amicroorganism comprises lysing the microorganism by using an enzyme.Preferred enzymes for lysing a microorganism are proteases andpolysaccharide-degrading enzymes such as hemicellulase (e.g.,hemicellulase from Aspergillus niger; Sigma Aldrich, St. Louis, Mo.;#H2125), pectinase (e.g., pectinase from Rhizopus sp.; Sigma Aldrich,St. Louis, Mo.; #P2401), Mannaway 4.0 L (Novozymes), cellulase (e.g.,cellulose from Trichoderma viride; Sigma Aldrich, St. Louis, Mo.;#C9422), and driselase (e.g., driselase from Basidiomycetes sp.; SigmaAldrich, St. Louis, Mo.; #D9515.

In other embodiments of the present invention, lysis is accomplishedusing an enzyme such as, for example, a cellulase such as apolysaccharide-degrading enzyme, optionally from Chlorella or aChlorella virus, or a proteases, such as Streptomyces griseus protease,chymotrypsin, proteinase K, proteases listed in Degradation ofPolylactide by Commercial Proteases, Oda Y et al., Journal of Polymersand the Environment, Volume 8, Number 1, January 2000, pp. 29-32(4),Alcalase 2.4 FG (Novozymes), and Flavourzyme 100 L (Novozymes). Anycombination of a protease and a polysaccharide-degrading enzyme can alsobe used, including any combination of the preceding proteases andpolysaccharide-degrading enzymes.

In another embodiment, lysis can be performed using an expeller press.In this process, biomass is forced through a screw-type device at highpressure, lysing the cells and causing the intracellular lipid to bereleased and separated from the protein and fiber (and other components)in the cell.

In another embodiment of the present invention, the step of lysing amicroorganism is performed by using ultrasound, i.e., sonication. Thus,cells can also by lysed with high frequency sound. The sound can beproduced electronically and transported through a metallic tip to anappropriately concentrated cellular suspension. This sonication (orultrasonication) disrupts cellular integrity based on the creation ofcavities in cell suspension.

In another embodiment of the present invention, the step of lysing amicroorganism is performed by mechanical lysis. Cells can be lysedmechanically and optionally homogenized to facilitate hydrocarbon (e.g.,lipid) collection. For example, a pressure disrupter can be used to pumpa cell containing slurry through a restricted orifice valve. Highpressure (up to 1500 bar) is applied, followed by an instant expansionthrough an exiting nozzle. Cell disruption is accomplished by threedifferent mechanisms: impingement on the valve, high liquid shear in theorifice, and sudden pressure drop upon discharge, causing an explosionof the cell. The method releases intracellular molecules. Alternatively,a ball mill can be used. In a ball mill, cells are agitated insuspension with small abrasive particles, such as beads. Cells breakbecause of shear forces, grinding between beads, and collisions withbeads. The beads disrupt the cells to release cellular contents. Cellscan also be disrupted by shear forces, such as with the use of blending(such as with a high speed or Waring blender as examples), the frenchpress, or even centrifugation in case of weak cell walls, to disruptcells.

In another embodiment of the present invention, the step of lysing amicroorganism is performed by applying an osmotic shock.

In another embodiment of the present invention, the step of lysing amicroorganism comprises infection of the microorganism with a lyticvirus. A wide variety of viruses are known to lyse microorganismssuitable for use in the present invention, and the selection and use ofa particular lytic virus for a particular microorganism is within thelevel of skill in the art. For example, paramecium bursaria chlorellavirus (PBCV-1) is the prototype of a group (family Phycodnaviridae,genus Chlorovirus) of large, icosahedral, plaque-forming,double-stranded DNA viruses that replicate in, and lyse, certainunicellular, eukaryotic chlorella-like green algae. Accordingly, anysusceptible microalgae can be lysed by infecting the culture with asuitable chlorella virus. Methods of infecting species of Chlorella witha chlorella virus are known. See for example Adv. Virus Res. 2006;66:293-336; Virology, 1999 Apr. 25; 257(1):15-23; Virology, 2004 Jan. 5;318(1):214-23; Nucleic Acids Symp. Ser. 2000; (44):161-2; J. Virol. 2006March; 80(5):2437-44; and Annu. Rev. Microbiol. 1999; 53:447-94.

In another embodiment of the present invention, the step of lysing amicroorganism comprises autolysis. In this embodiment, a microorganismaccording to the invention is genetically engineered to produce a lyticprotein that will lyse the microorganism. This lytic gene can beexpressed using an inducible promoter so that the cells can first begrown to a desirable density in a fermentor, followed by induction ofthe promoter to express the lytic gene to lyse the cells. In oneembodiment, the lytic gene encodes a polysaccharide-degrading enzyme. Incertain other embodiments, the lytic gene is a gene from a lytic virus.Thus, for example, a lytic gene from a Chlorella virus can be expressedin an algal cell; see Virology 260, 308-315 (1999); FEMS MicrobiologyLetters 180 (1999) 45-53; Virology 263, 376-387 (1999); and Virology230, 361-368 (1997). Expression of lytic genes is preferably done usingan inducible promoter, such as a promoter active in microalgae that isinduced by a stimulus such as the presence of a small molecule, light,heat, and other stimuli.

Various methods are available for separating lipids from cellularlysates produced by the above methods. For example, lipids and lipidderivatives such as fatty aldehydes, fatty alcohols, and hydrocarbonssuch as alkanes can be extracted with a hydrophobic solvent such ashexane (see Frenz et al. 1989, Enzyme Microb. Technol., 11:717). Lipidsand lipid derivatives can also be extracted using liquefaction (see forexample Sawayama et al. 1999, Biomass and Bioenergy 17:33-39 and Inoueet al. 1993, Biomass Bioenergy 6(4):269-274); oil liquefaction (see forexample Minowa et al. 1995, Fuel 74(12):1735-1738); and supercriticalCO₂ extraction (see for example Mendes et al. 2003, Inorganica ChimicaActa 356:328-334). Miao and Wu describe a protocol of the recovery ofmicroalgal lipid from a culture of Chlorella prototheocoides in whichthe cells were harvested by centrifugation, washed with distilled waterand dried by freeze drying. The resulting cell powder was pulverized ina mortar and then extracted with n-hexane. Miao and Wu, BiosourceTechnology (2006) 97:841-846.

Thus, lipids, lipid derivatives and hydrocarbons generated by themicroorganisms of the present invention can be recovered by extractionwith an organic solvent. In some cases, the preferred organic solvent ishexane. Typically, the organic solvent is added directly to the lysatewithout prior separation of the lysate components. In one embodiment,the lysate generated by one or more of the methods described above iscontacted with an organic solvent for a period of time sufficient toallow the lipid and/or hydrocarbon components to form a solution withthe organic solvent. In some cases, the solution can then be furtherrefined to recover specific desired lipid or hydrocarbon components.Hexane extraction methods are well known in the art.

Lipids and lipid derivatives such as fatty aldehydes, fatty alcohols,and hydrocarbons such as alkanes produced by cells as described hereincan be modified by the use of one or more enzymes, including a lipase,as described above. When the hydrocarbons are in the extracellularenvironment of the cells, the one or more enzymes can be added to thatenvironment under conditions in which the enzyme modifies thehydrocarbon or completes its synthesis from a hydrocarbon precursor.Alternatively, the hydrocarbons can be partially, or completely,isolated from the cellular material before addition of one or morecatalysts such as enzymes. Such catalysts are exogenously added, andtheir activity occurs outside the cell or in vitro.

Thus, lipids and hydrocarbons produced by cells in vivo, orenzymatically modified in vitro, as described herein can be optionallyfurther processed by conventional means. The processing can include“cracking” to reduce the size, and thus increase the hydrogen:carbonratio, of hydrocarbon molecules. Catalytic and thermal cracking methodsare routinely used in hydrocarbon and triglyceride oil processing.Catalytic methods involve the use of a catalyst, such as a solid acidcatalyst. The catalyst can be silica-alumina or a zeolite, which resultin the heterolytic, or asymmetric, breakage of a carbon-carbon bond toresult in a carbocation and a hydride anion. These reactiveintermediates then undergo either rearrangement or hydride transfer withanother hydrocarbon. The reactions can thus regenerate the intermediatesto result in a self-propagating chain mechanism. Hydrocarbons can alsobe processed to reduce, optionally to zero, the number of carbon-carbondouble, or triple, bonds therein. Hydrocarbons can also be processed toremove or eliminate a ring or cyclic structure therein. Hydrocarbons canalso be processed to increase the hydrogen:carbon ratio. This caninclude the addition of hydrogen (“hydrogenation”) and/or the “cracking”of hydrocarbons into smaller hydrocarbons.

Thermal methods involve the use of elevated temperature and pressure toreduce hydrocarbon size. An elevated temperature of about 800° C. andpressure of about 700 kPa can be used. These conditions generate“light,” a term that is sometimes used to refer to hydrogen-richhydrocarbon molecules (as distinguished from photon flux), while alsogenerating, by condensation, heavier hydrocarbon molecules which arerelatively depleted of hydrogen. The methodology provides homolytic, orsymmetrical, breakage and produces alkenes, which may be optionallyenzymatically saturated as described above.

Catalytic and thermal methods are standard in plants for hydrocarbonprocessing and oil refining. Thus hydrocarbons produced by cells asdescribed herein can be collected and processed or refined viaconventional means. See Hillen et al. (Biotechnology and Bioengineering,Vol. XXIV:193-205 (1982)) for a report on hydrocracking ofmicroalgae-produced hydrocarbons. In alternative embodiments, thefraction is treated with another catalyst, such as an organic compound,heat, and/or an inorganic compound. For processing of lipids intobiodiesel, a transesterification process is used as described below inthis Section.

Hydrocarbons produced via methods of the present invention are useful ina variety of industrial applications. For example, the production oflinear alkylbenzene sulfonate (LAS), an anionic surfactant used innearly all types of detergents and cleaning preparations, utilizeshydrocarbons generally comprising a chain of 10-14 carbon atoms. See,for example, U.S. Pat. Nos. 6,946,430; 5,506,201; 6,692,730; 6,268,517;6,020,509; 6,140,302; 5,080,848; and 5,567,359. Surfactants, such asLAS, can be used in the manfacture of personal care compositions anddetergents, such as those described in U.S. Pat. Nos. 5,942,479;6,086,903; 5,833,999; 6,468,955; and 6,407,044.

Increasing interest is directed to the use of hydrocarbon components ofbiological origin in fuels, such as biodiesel, renewable diesel, and jetfuel, since renewable biological starting materials that may replacestarting materials derived from fossil fuels are available, and the usethereof is desirable. There is an urgent need for methods for producinghydrocarbon components from biological materials. The present inventionfulfills this need by providing methods for production of biodiesel,renewable diesel, and jet fuel using the lipids generated by the methodsdescribed herein as a biological material to produce biodiesel,renewable diesel, and jet fuel.

Traditional diesel fuels are petroleum distillates rich in paraffinichydrocarbons. They have boiling ranges as broad as 370° to 780° F.,which are suitable for combustion in a compression ignition engine, suchas a diesel engine vehicle. The American Society of Testing andMaterials (ASTM) establishes the grade of diesel according to theboiling range, along with allowable ranges of other fuel properties,such as cetane number, cloud point, flash point, viscosity, anilinepoint, sulfur content, water content, ash content, copper stripcorrosion, and carbon residue. Technically, any hydrocarbon distillatematerial derived from biomass or otherwise that meets the appropriateASTM specification can be defined as diesel fuel (ASTM D975), jet fuel(ASTM D1655), or as biodiesel if it is a fatty acid methyl ester (ASTMD6751).

After extraction, lipid and/or hydrocarbon components recovered from themicrobial biomass described herein can be subjected to chemicaltreatment to manufacture a fuel for use in diesel vehicles and jetengines.

Biodiesel is a liquid which varies in color—between golden and darkbrown—depending on the production feedstock. It is practicallyimmiscible with water, has a high boiling point and low vapor pressure.Biodiesel refers to a diesel-equivalent processed fuel for use indiesel-engine vehicles. Biodiesel is biodegradable and non-toxic. Anadditional benefit of biodiesel over conventional diesel fuel is lowerengine wear. Typically, biodiesel comprises C14-C18 alkyl esters.Various processes convert biomass or a lipid produced and isolated asdescribed herein to diesel fuels. A preferred method to producebiodiesel is by transesterification of a lipid as described herein. Apreferred alkyl ester for use as biodiesel is a methyl ester or ethylester.

Biodiesel produced by a method described herein can be used alone orblended with conventional diesel fuel at any concentration in mostmodern diesel-engine vehicles. When blended with conventional dieselfuel (petroleum diesel), biodiesel may be present from about 0.1% toabout 99.9%. Much of the world uses a system known as the “B” factor tostate the amount of biodiesel in any fuel mix. For example, fuelcontaining 20% biodiesel is labeled B20. Pure biodiesel is referred toas B 100.

Biodiesel can also be used as a heating fuel in domestic and commercialboilers. Existing oil boilers may contain rubber parts and may requireconversion to run on biodiesel. The conversion process is usuallyrelatively simple, involving the exchange of rubber parts for syntheticparts due to biodiesel being a strong solvent. Due to its strong solventpower, burning biodiesel will increase the efficiency of boilers.Biodiesel can be used as an additive in formulations of diesel toincrease the lubricity of pure Ultra-Low Sulfur Diesel (ULSD) fuel,which is advantageous because it has virtually no sulfur content.Biodiesel is a better solvent than petrodiesel and can be used to breakdown deposits of residues in the fuel lines of vehicles that havepreviously been run on petrodiesel.

Biodiesel can be produced by transesterification of triglyceridescontained in oil-rich biomass. Thus, in another aspect of the presentinvention a method for producing biodiesel is provided. In a preferredembodiment, the method for producing biodiesel comprises the steps of(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing a lipid-containing microorganism to produce a lysate,(c) isolating lipid from the lysed microorganism, and (d)transesterifying the lipid composition, whereby biodiesel is produced.Methods for growth of a microorganism, lysing a microorganism to producea lysate, treating the lysate in a medium comprising an organic solventto form a heterogeneous mixture and separating the treated lysate into alipid composition have been described above and can also be used in themethod of producing biodiesel.

The lipid profile of the biodiesel is usually highly similar to thelipid profile of the feedstock oil. Other oils provided by the methodsand compositions of the invention can be subjected totransesterification to yield biodiesel with lipid profiles including (a)at least 1%-5%, preferably at least 4%, C8-C14; (b) at least 0.25%-1%,preferably at least 0.3%, C8; (c) at least 1%-5%, preferably at least2%, C10; (d) at least 1%-5%, preferably at least 2%, C12; and (3) atleast 20%-40%, preferably at least 30%, C8-C14.

Lipid compositions can be subjected to transesterification to yieldlong-chain fatty acid esters useful as biodiesel. Preferredtransesterification reactions are outlined below and include basecatalyzed transesterification and transesterification using recombinantlipases. In a base-catalyzed transesterification process, thetriacylglycerides are reacted with an alcohol, such as methanol orethanol, in the presence of an alkaline catalyst, typically potassiumhydroxide. This reaction forms methyl or ethyl esters and glycerin(glycerol) as a byproduct.

Animal and plant oils are typically made of triglycerides which areesters of free fatty acids with the trihydric alcohol, glycerol. Intransesterification, the glycerol in a triacylglyceride (TAG) isreplaced with a short-chain alcohol such as methanol or ethanol. Atypical reaction scheme is as follows:

In this reaction, the alcohol is deprotonated with a base to make it astronger nucleophile. Commonly, ethanol or methanol is used in vastexcess (up to 50-fold). Normally, this reaction will proceed eitherexceedingly slowly or not at all. Heat, as well as an acid or base canbe used to help the reaction proceed more quickly. The acid or base arenot consumed by the transesterification reaction, thus they are notreactants but catalysts. Almost all biodiesel has been produced usingthe base-catalyzed technique as it requires only low temperatures andpressures and produces over 98% conversion yield (provided the startingoil is low in moisture and free fatty acids).

Transesterification has also been carried out, as discussed above, usingan enzyme, such as a lipase instead of a base. Lipase-catalyzedtransesterification can be carried out, for example, at a temperaturebetween the room temperature and 80° C., and a mole ratio of the TAG tothe lower alcohol of greater than 1:1, preferably about 3:1. Lipasessuitable for use in transesterification include, but are not limited to,those listed in Table 9. Other examples of lipases useful fortransesterification are found in, e.g. U.S. Pat. Nos. 4,798,793;4,940,845 5,156,963; 5,342,768; 5,776,741 and WO89/01032. Such lipasesinclude, but are not limited to, lipases produced by microorganisms ofRhizopus, Aspergillus, Candida, Mucor, Pseudomonas, Rhizomucor, Candida,and Humicola and pancreas lipase.

TABLE 9 Lipases suitable for use in transesterification. Aspergillusniger lipase ABG73614, Candida antarctica lipase B (novozym-435)CAA83122, Candida cylindracea lipase AAR24090, Candida lipolytica lipase(Lipase L; Amano Pharmaceutical Co., Ltd.), Candida rugosa lipase (e.g.,Lipase-OF; Meito Sangyo Co., Ltd.), Mucor miehei lipase (Lipozyme IM20), Pseudomonas fluorescens lipase AAA25882, Rhizopus japonicas lipase(Lilipase A-10FG) Q7M4U7_1, Rhizomucor miehei lipase B34959, Rhizopusoryzae lipase (Lipase F) AAF32408, Serratia marcescens lipase (SMEnzyme) ABI13521, Thermomyces lanuginosa lipase CAB58509, Lipase P(Nagase ChemteX Corporation), and Lipase QLM (Meito Sangyo Co., Ltd.,Nagoya, Japan)

One challenge to using a lipase for the production of fatty acid esterssuitable for biodiesel is that the price of lipase is much higher thanthe price of sodium hydroxide (NaOH) used by the strong base process.This challenge has been addressed by using an immobilized lipase, whichcan be recycled. However, the activity of the immobilized lipase must bemaintained after being recycled for a minimum number of cycles to allowa lipase-based process to compete with the strong base process in termsof the production cost. Immobilized lipases are subject to poisoning bythe lower alcohols typically used in transesterification. U.S. Pat. No.6,398,707 (issued Jun. 4, 2002 to Wu et al.) describes methods forenhancing the activity of immobilized lipases and regeneratingimmobilized lipases having reduced activity. Some suitable methodsinclude immersing an immobilized lipase in an alcohol having a carbonatom number not less than 3 for a period of time, preferably from 0.5-48hours, and more preferably from 0.5-1.5 hours. Some suitable methodsalso include washing a deactivated immobilized lipase with an alcoholhaving a carbon atom number not less than 3 and then immersing thedeactivated immobilized lipase in a vegetable oil for 0.5-48 hours.

In particular embodiments, a recombinant lipase is expressed in the samemicroorganisms that produce the lipid on which the lipase acts. Suitablerecombinant lipases include those listed above in Table 9 and/or havingGenBank Accession numbers listed above in Table 9, or a polypeptide thathas at least 70% amino acid identity with one of the lipases listedabove in Table 9 and that exhibits lipase activity. In additionalembodiments, the enzymatic activity is present in a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% identity with one of theabove described sequences, all of which are hereby incorporated byreference as if fully set forth. DNA encoding the lipase and selectablemarker is preferably codon-optimized cDNA. Methods of recoding genes forexpression in microalgae are described in U.S. Pat. No. 7,135,290.

The common international standard for biodiesel is EN 14214. ASTM D6751is the most common biodiesel standard referenced in the United Statesand Canada. Germany uses DIN EN 14214 and the UK requires compliancewith BS EN 14214. Basic industrial tests to determine whether theproducts conform to these standards typically include gaschromatography, HPLC, and others. Biodiesel meeting the qualitystandards is very non-toxic, with a toxicity rating (LD₅₀) of greaterthan 50 mL/kg.

Although biodiesel that meets the ASTM standards has to be non-toxic,there can be contaminants which tend to crystallize and/or precipitateand fall out of solution as sediment. Sediment formation is particularlya problem when biodiesel is used at lower temperatures. The sediment orprecipitates may cause problems such as decreasing fuel flow, cloggingfuel lines, clogging filters, etc. Processes are well-known in the artthat specifically deal with the removal of these contaminants andsediments in biodiesel in order to produce a higher quality product.Examples for such processes include, but are not limited to,pretreatment of the oil to remove contaiminants such as phospholipidsand free fatty acids (e.g., degumming, caustic refining and silicaadsorbant filtration) and cold filtration. Cold filtration is a processthat was developed specifically to remove any particulates and sedimentsthat are present in the biodiesel after production. This process coolsthe biodiesel and filters out any sediments or precipitates that mightform when the fuel is used at a lower temperature. Such a process iswell known in the art and is described in US Patent ApplicationPublication No. 2007-0175091. Suitable methods may include cooling thebiodiesel to a temperature of less than about 38° C. so that theimpurities and contaminants precipitate out as particulates in thebiodiesel liquid. Diatomaceous earth or other filtering material maythen added to the cooled biodiesel to form a slurry, which may thenfiltered through a pressure leaf or other type of filter to remove theparticulates. The filtered biodiesel may then be run through a polishfilter to remove any remaining sediments and diatomaceous earth, so asto produce the final biodiesel product.

Example 13 describes the production of biodiesel using triglyceride oilfrom Prototheca moriformis. The Cold Soak Filterability by the ASTMD6751 A1 method of the biodiesel produced in Example 13 was 120 secondsfor a volume of 300 ml. This test involves filtration of 300 ml of B100, chilled to 40° F. for 16 hours, allowed to warm to room temp, andfiltered under vacuum using 0.7 micron glass fiber filter with stainlesssteel support. Oils of the invention can be transesterified to generatebiodiesel with a cold soak time of less than 120 seconds, less than 100seconds, and less than 90 seconds.

Subsequent processes may also be used if the biodiesel will be used inparticularly cold temperatures. Such processes include winterization andfractionation. Both processes are designed to improve the cold flow andwinter performance of the fuel by lowering the cloud point (thetemperature at which the biodiesel starts to crystallize). There areseveral approaches to winterizing biodiesel. One approach is to blendthe biodiesel with petroleum diesel. Another approach is to useadditives that can lower the cloud point of biodiesel. Another approachis to remove saturated methyl esters indiscriminately by mixing inadditives and allowing for the crystallization of saturates and thenfiltering out the crystals. Fractionation selectively separates methylesters into individual components or fractions, allowing for the removalor inclusion of specific methyl esters. Fractionation methods includeurea fractionation, solvent fractionation and thermal distillation.

Another valuable fuel provided by the methods of the present inventionis renewable diesel, which comprises alkanes, such as C10:0, C12:0,C14:0, C16:0 and C18:0 and thus, are distinguishable from biodiesel.High quality renewable diesel conforms to the ASTM D975 standard. Thelipids produced by the methods of the present invention can serve asfeedstock to produce renewable diesel. Thus, in another aspect of thepresent invention, a method for producing renewable diesel is provided.Renewable diesel can be produced by at least three processes:hydrothermal processing (hydrotreating); hydroprocessing; and indirectliquefaction. These processes yield non-ester distillates. During theseprocesses, triacylglycerides produced and isolated as described herein,are converted to alkanes.

In one embodiment, the method for producing renewable diesel comprises(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing the microorganism to produce a lysate, (c) isolatinglipid from the lysed microorganism, and (d) deoxygenating andhydrotreating the lipid to produce an alkane, whereby renewable dieselis produced. Lipids suitable for manufacturing renewable diesel can beobtained via extraction from microbial biomass using an organic solventsuch as hexane, or via other methods, such as those described in U.S.Pat. No. 5,928,696. Some suitable methods may include mechanicalpressing and centrifuging.

In some methods, the microbial lipid is first cracked in conjunctionwith hydrotreating to reduce carbon chain length and saturate doublebonds, respectively. The material is then isomerized, also inconjunction with hydrotreating. The naptha fraction can then be removedthrough distillation, followed by additional distillation to vaporizeand distill components desired in the diesel fuel to meet an ASTM D975standard while leaving components that are heavier than desired formeeting the D975 standard. Hydrotreating, hydrocracking, deoxygenationand isomerization methods of chemically modifying oils, includingtriglyceride oils, are well known in the art. See for example Europeanpatent applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;5,885,440; 6,881,873.

In one embodiment of the method for producing renewable diesel, treatingthe lipid to produce an alkane is performed by hydrotreating of thelipid composition. In hydrothermal processing, typically, biomass isreacted in water at an elevated temperature and pressure to form oilsand residual solids. Conversion temperatures are typically 300° to 660°F., with pressure sufficient to keep the water primarily as a liquid,100 to 170 standard atmosphere (atm). Reaction times are on the order of15 to 30 minutes. After the reaction is completed, the organics areseparated from the water. Thereby a distillate suitable for diesel isproduced.

In some methods of making renewable diesel, the first step of treating atriglyceride is hydroprocessing to saturate double bonds, followed bydeoxygenation at elevated temperature in the presence of hydrogen and acatalyst. In some methods, hydrogenation and deoxygenation occur in thesame reaction. In other methods deoxygenation occurs beforehydrogenation. Isomerization is then optionally performed, also in thepresence of hydrogen and a catalyst. Naphtha components are preferablyremoved through distillation. For examples, see U.S. Pat. No. 5,475,160(hydrogenation of triglycerides); U.S. Pat. No. 5,091,116(deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815(hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).

One suitable method for the hydrogenation of triglycerides includespreparing an aqueous solution of copper, zinc, magnesium and lanthanumsalts and another solution of alkali metal or preferably, ammoniumcarbonate. The two solutions may be heated to a temperature of about 20°C. to about 85° C. and metered together into a precipitation containerat rates such that the pH in the precipitation container is maintainedbetween 5.5 and 7.5 in order to form a catalyst. Additional water may beused either initially in the precipitation container or addedconcurrently with the salt solution and precipitation solution. Theresulting precipitate may then be thoroughly washed, dried, calcined atabout 300° C. and activated in hydrogen at temperatures ranging fromabout 100° C. to about 400° C. One or more triglycerides may then becontacted and reacted with hydrogen in the presence of theabove-described catalyst in a reactor. The reactor may be a trickle bedreactor, fixed bed gas-solid reactor, packed bubble column reactor,continuously stirred tank reactor, a slurry phase reactor, or any othersuitable reactor type known in the art. The process may be carried outeither batchwise or in continuous fashion. Reaction temperatures aretypically in the range of from about 170° C. to about 250° C. whilereaction pressures are typically in the range of from about 300 psig toabout 2000 psig. Moreover, the molar ratio of hydrogen to triglyceridein the process of the present invention is typically in the range offrom about 20:1 to about 700:1. The process is typically carried out ata weight hourly space velocity (WHSV) in the range of from about 0.1hr⁻¹ to about 5 hr⁻¹. One skilled in the art will recognize that thetime period required for reaction will vary according to the temperatureused, the molar ratio of hydrogen to triglyceride, and the partialpressure of hydrogen. The products produced by the such hydrogenationprocesses include fatty alcohols, glycerol, traces of paraffins andunreacted triglycerides. These products are typically separated byconventional means such as, for example, distillation, extraction,filtration, crystallization, and the like.

Petroleum refiners use hydroprocessing to remove impurities by treatingfeeds with hydrogen. Hydroprocessing conversion temperatures aretypically 300° to 700° F. Pressures are typically 40 to 100 atm. Thereaction times are typically on the order of 10 to 60 minutes. Solidcatalysts are employed to increase certain reaction rates, improveselectivity for certain products, and optimize hydrogen consumption.

Suitable methods for the deoxygenation of an oil includes heating an oilto a temperature in the range of from about 350° F. to about 550° F. andcontinuously contacting the heated oil with nitrogen under at leastpressure ranging from about atmospeheric to above for at least about 5minutes.

Suitable methods for isomerization include using alkali isomerizationand other oil isomerization known in the art.

Hydrotreating and hydroprocessing ultimately lead to a reduction in themolecular weight of the triglyceride feed. The triglyceride molecule isreduced to four hydrocarbon molecules under hydroprocessing conditions:a propane molecule and three heavier hydrocarbon molecules, typically inthe C8 to C18 range.

Thus, in one embodiment, the product of one or more chemical reaction(s)performed on lipid compositions of the invention is an alkane mixturethat comprises ASTM D975 renewable diesel. Production of hydrocarbons bymicroorganisms is reviewed by Metzger et al. Appl Microbiol Biotechnol(2005) 66: 486-496 and A Look Back at the U.S. Department of Energy'sAquatic Species Program: Biodiesel from Algae, NREL/TP-580-24190, JohnSheehan, Terri Dunahay, John Benemann and Paul Roessler (1998).

The distillation properties of a diesel fuel is described in terms ofT10-T90 (temperature at 10% and 90%, respectively, volume distilled).Renewable diesel was produced from Prototheca moriformis triglycerideoil and is described in Example 13. The T10-T90 of the material producedin Example 13 was 57.9° C. Methods of hydrotreating, isomerization, andother covalent modification of oils disclosed herein, as well as methodsof distillation and fractionation (such as cold filtration) disclosedherein, can be employed to generate renewable diesel compositions withother T10-T90 ranges, such as 20, 25, 30, 35, 40, 45, 50, 60 and 65° C.using triglyceride oils produced according to the methods disclosedherein.

The T10 of the material produced in Example 13 was 242.1° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other T10 values, such as T10 between180 and 295, between 190 and 270, between 210 and 250, between 225 and245, and at least 290.

The T90 of the material produced in Example 13 was 300° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein can be employed to generaterenewable diesel compositions with other T90 values, such as T90 between280 and 380, between 290 and 360, between 300 and 350, between 310 and340, and at least 290.

The FBP of the material produced in Example 13 was 300° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other FBP values, such as FBP between290 and 400, between 300 and 385, between 310 and 370, between 315 and360, and at least 300.

Other oils provided by the methods and compositions of the invention canbe subjected to combinations of hydrotreating, isomerization, and othercovalent modification including oils with lipid profiles including (a)at least 1%-5%, preferably at least 4%, C8-C14; (b) at least 0.25%-1%,preferably at least 0.3%, C8; (c) at least 1%-5%, preferably at least2%, C10; (d) at least 1%-5%, preferably at least 2%, C12; and (3) atleast 20%-40%, preferably at least 30% C8-C14.

A traditional ultra-low sulfur diesel can be produced from any form ofbiomass by a two-step process. First, the biomass is converted to asyngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then,the syngas is catalytically converted to liquids. Typically, theproduction of liquids is accomplished using Fischer-Tropsch (FT)synthesis. This technology applies to coal, natural gas, and heavy oils.Thus, in yet another preferred embodiment of the method for producingrenewable diesel, treating the lipid composition to produce an alkane isperformed by indirect liquefaction of the lipid composition.

The present invention also provides methods to produce jet fuel. Jetfuel is clear to straw colored. The most common fuel is anunleaded/paraffin oil-based fuel classified as Aeroplane A-1, which isproduced to an internationally standardized set of specifications. Jetfuel is a mixture of a large number of different hydrocarbons, possiblyas many as a thousand or more. The range of their sizes (molecularweights or carbon numbers) is restricted by the requirements for theproduct, for example, freezing point or smoke point. Kerosone-typeAeroplane fuel (including Jet A and Jet A-1) has a carbon numberdistribution between about 8 and 16 carbon numbers. Wide-cut ornaphta-type Aeroplane fuel (including Jet B) typically has a carbonnumber distribution between about 5 and 15 carbons.

Both Aeroplanes (Jet A and Jet B) may contain a number of additives.Useful additives include, but are not limited to, antioxidants,antistatic agents, corrosion inhibitors, and fuel system icing inhibitor(FSII) agents. Antioxidants prevent gumming and usually, are based onalkylated phenols, for example, AO-30, AO-31, or AO-37. Antistaticagents dissipate static electricity and prevent sparking. Stadis 450with dinonylnaphthylsulfonic acid (DINNSA) as the active ingredient, isan example. Corrosion inhibitors, e.g., DCI-4A is used for civilian andmilitary fuels and DCI-6A is used for military fuels. FSII agents,include, e.g., Di-EGME.

In one embodiment of the invention, a jet fuel is produced by blendingalgal fuels with existing jet fuel. The lipids produced by the methodsof the present invention can serve as feedstock to produce jet fuel.Thus, in another aspect of the present invention, a method for producingjet fuel is provided. Herewith two methods for producing jet fuel fromthe lipids produced by the methods of the present invention areprovided: fluid catalytic cracking (FCC); and hydrodeoxygenation (HDO).

Fluid Catalytic Cracking (FCC) is one method which is used to produceolefins, especially propylene from heavy crude fractions. The lipidsproduced by the method of the present invention can be converted toolefins. The process involves flowing the lipids produced through an FCCzone and collecting a product stream comprised of olefins, which isuseful as a jet fuel. The lipids produced are contacted with a crackingcatalyst at cracking conditions to provide a product stream comprisingolefins and hydrocarbons useful as jet fuel.

In one embodiment, the method for producing jet fuel comprises (a)cultivating a lipid-containing microorganism using methods disclosedherein, (b) lysing the lipid-containing microorganism to produce alysate, (c) isolating lipid from the lysate, and (d) treating the lipidcomposition, whereby jet fuel is produced. In one embodiment of themethod for producing a jet fuel, the lipid composition can be flowedthrough a fluid catalytic cracking zone, which, in one embodiment, maycomprise contacting the lipid composition with a cracking catalyst atcracking conditions to provide a product stream comprising C₂-C₅olefins.

In certain embodiments of this method, it may be desirable to remove anycontaminants that may be present in the lipid composition. Thus, priorto flowing the lipid composition through a fluid catalytic crackingzone, the lipid composition is pretreated. Pretreatment may involvecontacting the lipid composition with an ion-exchange resin. The ionexchange resin is an acidic ion exchange resin, such as Amberlyst™-15and can be used as a bed in a reactor through which the lipidcomposition is flowed, either upflow or downflow. Other pretreatmentsmay include mild acid washes by contacting the lipid composition with anacid, such as sulfuric, acetic, nitric, or hydrochloric acid. Contactingis done with a dilute acid solution usually at ambient temperature andatmospheric pressure.

The lipid composition, optionally pretreated, is flowed to an FCC zonewhere the hydrocarbonaceous components are cracked to olefins. Catalyticcracking is accomplished by contacting the lipid composition in areaction zone with a catalyst composed of finely divided particulatematerial. The reaction is catalytic cracking, as opposed tohydrocracking, and is carried out in the absence of added hydrogen orthe consumption of hydrogen. As the cracking reaction proceeds,substantial amounts of coke are deposited on the catalyst. The catalystis regenerated at high temperatures by burning coke from the catalyst ina regeneration zone. Coke-containing catalyst, referred to herein as“coked catalyst”, is continually transported from the reaction zone tothe regeneration zone to be regenerated and replaced by essentiallycoke-free regenerated catalyst from the regeneration zone. Fluidizationof the catalyst particles by various gaseous streams allows thetransport of catalyst between the reaction zone and regeneration zone.Methods for cracking hydrocarbons, such as those of the lipidcomposition described herein, in a fluidized stream of catalyst,transporting catalyst between reaction and regeneration zones, andcombusting coke in the regenerator are well known by those skilled inthe art of FCC processes. Exemplary FCC applications and catalystsuseful for cracking the lipid composition to produce C₂-C₅ olefins aredescribed in U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporatedin their entirety by reference.

Suitable FCC catalysts generally comprise at least two components thatmay or may not be on the same matrix. In some embodiments, both twocomponents may be circulated throughout the entire reaction vessel. Thefirst component generally includes any of the well-known catalysts thatare used in the art of fluidized catalytic cracking, such as an activeamorphous clay-type catalyst and/or a high activity, crystallinemolecular sieve. Molecular sieve catalysts may be preferred overamorphous catalysts because of their much-improved selectivity todesired products. IN some preferred embodiments, zeolites may be used asthe molecular sieve in the FCC processes. Preferably, the first catalystcomponent comprises a large pore zeolite, such as an Y-type zeolite, anactive alumina material, a binder material, comprising either silica oralumina and an inert filler such as kaolin.

In one embodiment, cracking the lipid composition of the presentinvention, takes place in the riser section or, alternatively, the liftsection, of the FCC zone. The lipid composition is introduced into theriser by a nozzle resulting in the rapid vaporization of the lipidcomposition. Before contacting the catalyst, the lipid composition willordinarily have a temperature of about 149° C. to about 316° C. (300° F.to 600° F.). The catalyst is flowed from a blending vessel to the riserwhere it contacts the lipid composition for a time of abort 2 seconds orless.

The blended catalyst and reacted lipid composition vapors are thendischarged from the top of the riser through an outlet and separatedinto a cracked product vapor stream including olefins and a collectionof catalyst particles covered with substantial quantities of coke andgenerally referred to as “coked catalyst.” In an effort to minimize thecontact time of the lipid composition and the catalyst which may promotefurther conversion of desired products to undesirable other products,any arrangement of separators such as a swirl arm arrangement can beused to remove coked catalyst from the product stream quickly. Theseparator, e.g. swirl arm separator, is located in an upper portion of achamber with a stripping zone situated in the lower portion of thechamber. Catalyst separated by the swirl arm arrangement drops down intothe stripping zone. The cracked product vapor stream comprising crackedhydrocarbons including light olefins and some catalyst exit the chambervia a conduit which is in communication with cyclones. The cyclonesremove remaining catalyst particles from the product vapor stream toreduce particle concentrations to very low levels. The product vaporstream then exits the top of the separating vessel. Catalyst separatedby the cyclones is returned to the separating vessel and then to thestripping zone. The stripping zone removes adsorbed hydrocarbons fromthe surface of the catalyst by counter-current contact with steam.

Low hydrocarbon partial pressure operates to favor the production oflight olefins. Accordingly, the riser pressure is set at about 172 to241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressureof about 69 to 138 kPa (10 to 20 psia). This relatively low partialpressure for hydrocarbon is achieved by using steam as a diluent to theextent that the diluent is 10 to 55 wt-% of lipid composition andpreferably about 15 wt-% of lipid composition. Other diluents such asdry gas can be used to reach equivalent hydrocarbon partial pressures.

The temperature of the cracked stream at the riser outlet will be about510° C. to 621° C. (950° F. to 1150° F.). However, riser outlettemperatures above 566° C. (1050° F.) make more dry gas and moreolefins. Whereas, riser outlet temperatures below 566° C. (1050° F.)make less ethylene and propylene. Accordingly, it is preferred to runthe FCC process at a preferred temperature of about 566° C. to about630° C., preferred pressure of about 138 kPa to about 240 kPa (20 to 35psia). Another condition for the process is the catalyst to lipidcomposition ratio which can vary from about 5 to about 20 and preferablyfrom about 10 to about 15.

In one embodiment of the method for producing a jet fuel, the lipidcomposition is introduced into the lift section of an FCC reactor. Thetemperature in the lift section will be very hot and range from about700° C. (1292° F.) to about 760° C. (1400° F.) with a catalyst to lipidcomposition ratio of about 100 to about 150. It is anticipated thatintroducing the lipid composition into the lift section will produceconsiderable amounts of propylene and ethylene.

In another embodiment of the method for producing a jet fuel using thelipid composition or the lipids produced as described herein, thestructure of the lipid composition or the lipids is broken by a processreferred to as hydrodeoxygenation (HDO). HDO means removal of oxygen bymeans of hydrogen, that is, oxygen is removed while breaking thestructure of the material. Olefinic double bonds are hydrogenated andany sulphur and nitrogen compounds are removed. Sulphur removal iscalled hydrodesulphurization (HDS). Pretreatment and purity of the rawmaterials (lipid composition or the lipids) contribute to the servicelife of the catalyst.

Generally in the HDO/HDS step, hydrogen is mixed with the feed stock(lipid composition or the lipids) and then the mixture is passed througha catalyst bed as a co-current flow, either as a single phase or a twophase feed stock. After the HDO/MDS step, the product fraction isseparated and passed to a separate isomerzation reactor. Anisomerization reactor for biological starting material is described inthe literature (FI 100 248) as a co-current reactor.

The process for producing a fuel by hydrogenating a hydrocarbon feed,e.g., the lipid composition or the lipids herein, can also be performedby passing the lipid composition or the lipids as a co-current flow withhydrogen gas through a first hydrogenation zone, and thereafter thehydrocarbon effluent is further hydrogenated in a second hydrogenationzone by passing hydrogen gas to the second hydrogenation zone as acounter-current flow relative to the hydrocarbon effluent. Exemplary HDOapplications and catalysts useful for cracking the lipid composition toproduce C₂-C₅ olefins are described in U.S. Pat. No. 7,232,935, which isincorporated in its entirety by reference.

Typically, in the hydrodeoxygenation step, the structure of thebiological component, such as the lipid composition or lipids herein, isdecomposed, oxygen, nitrogen, phosphorus and sulphur compounds, andlight hydrocarbons as gas are removed, and the olefinic bonds arehydrogenated. In the second step of the process, i.e. in the so-calledisomerization step, isomerzation is carried out for branching thehydrocarbon chain and improving the performance of the paraffin at lowtemperatures.

In the first step, i.e. HDO step, of the cracking process, hydrogen gasand the lipid composition or lipids herein which are to be hydrogenatedare passed to a HDO catalyst bed system either as co-current orcounter-current flows, said catalyst bed system comprising one or morecatalyst bed(s), preferably 1-3 catalyst beds. The HDO step is typicallyoperated in a co-current manner. In case of a HDO catalyst bed systemcomprising two or more catalyst beds, one or more of the beds may beoperated using the counter-current flow principle. In the HDO step, thepressure varies between 20 and 150 bar, preferably between 50 and 100bar, and the temperature varies between 200 and 500° C., preferably inthe range of 300-400° C. In the HDO step, known hydrogenation catalystscontaining metals from Group VII and/or VIB of the Periodic System maybe used. Preferably, the hydrogenation catalysts are supported Pd, Pt,Ni, NiMo or a CoMo catalysts, the support being alumina and/or silica.Typically, NiMo/Al₂O₃ and CoMo/Al₂O₃ catalysts are used.

Prior to the HDO step, the lipid composition or lipids herein mayoptionally be treated by prehydrogenation under milder conditions thusavoiding side reactions of the double bonds. Such prehydrogenation iscarried out in the presence of a prehydrogenation catalyst attemperatures of 50-400° C. and at hydrogen pressures of 1-200 bar,preferably at a temperature between 150 and 250° C. and at a hydrogenpressure between 10 and 100 bar. The catalyst may contain metals fromGroup VIII and/or VIB of the Periodic System. Preferably, theprehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMocatalyst, the support being alumina and/or silica.

A gaseous stream from the HDO step containing hydrogen is cooled andthen carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulphurcompounds, gaseous light hydrocarbons and other impurities are removedtherefrom. After compressing, the purified hydrogen or recycled hydrogenis returned back to the first catalyst bed and/or between the catalystbeds to make up for the withdrawn gas stream. Water is removed from thecondensed liquid. The liquid is passed to the first catalyst bed orbetween the catalyst beds.

After the HDO step, the product is subjected to an isomerization step.It is substantial for the process that the impurities are removed ascompletely as possible before the hydrocarbons are contacted with theisomerization catalyst. The isomerization step comprises an optionalstripping step, wherein the reaction product from the HDO step may bepurified by stripping with water vapour or a suitable gas such as lighthydrocarbon, nitrogen or hydrogen. The optional stripping step iscarried out in counter-current manner in a unit upstream of theisomerization catalyst, wherein the gas and liquid are contacted witheach other, or before the actual isomerization reactor in a separatestripping unit utilizing counter-current principle.

After the stripping step the hydrogen gas and the hydrogenated lipidcomposition or lipids herein, and optionally an n-paraffin mixture, arepassed to a reactive isomerization unit comprising one or severalcatalyst bed(s). The catalyst beds of the isomerization step may operateeither in co-current or counter-current manner.

It is important for the process that the counter-current flow principleis applied in the isomerization step. In the isomerization step this isdone by carrying out either the optional stripping step or theisomerization reaction step or both in counter-current manner. In theisomerzation step, the pressure varies in the range of 20-150 bar,preferably in the range of 20-100 bar, the temperature being between 200and 500° C., preferably between 300 and 400° C. In the isomerizationstep, isomerization catalysts known in the art may be used. Suitableisomerization catalysts contain molecular sieve and/or a metal fromGroup VII and/or a carrier. Preferably, the isomerization catalystcontains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrierite and Pt, Pdor Ni and Al₂O₃ or SiO₂. Typical isomerization catalysts are, forexample, Pt/SAPO-11/Al₂O₃, Pt/ZSM-22/Al₂O₃, Pt/ZSM-23/Al₂O₃ andPt/SAPO-11/SiO₂. The isomerization step and the HDO step may be carriedout in the same pressure vessel or in separate pressure vessels.Optional prehydrogenation may be carried out in a separate pressurevessel or in the same pressure vessel as the HDO and isomerizationsteps.

Thus, in one embodiment, the product of one or more chemical reactionsis an alkane mixture that comprises HRJ-5. In another embodiment, theproduct of the one or more chemical reactions is an alkane mixture thatcomprises ASTM D1655 jet fuel. In some embodiments, the compositioncomforming to the specification of ASTM 1655 jet fuel has a sulfurcontent that is less than 10 ppm. In other embodiments, the compositionconforming to the specification of ASTM 1655 jet fuel has a T10 value ofthe distillation curve of less than 205° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has afinal boiling point (FBP) of less than 300° C. In another embodiment,the composition conforming to the specification of ASTM 1655 jet fuelhas a flash point of at least 38° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has adensity between 775K/M³ and 840K/M³. In yet another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has afreezing point that is below −47° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has anet Heat of Combustion that is at least 42.8 MJ/K. In anotherembodiment, the composition conforming to the specification of ASTM 1655jet fuel has a hydrogen content that is at least 13.4 mass %. In anotherembodiment, the composition conforming to the specification of ASTM 1655jet fuel has a thermal stability, as tested by quantitative gravimetricJFTOT at 260° C., that is below 3 mm of Hg. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has anexistent gum that is below 7 mg/dl.

Thus, the present invention discloses a variety of methods in whichchemical modification of microalgal lipid is undertaken to yieldproducts useful in a variety of industrial and other applications.Examples of processes for modifying oil produced by the methodsdisclosed herein include, but are not limited to, hydrolysis of the oil,hydroprocessing of the oil, and esterification of the oil. Otherchemical modification of microalgal lipid include, without limitation,epoxidation, oxidation, hydrolysis, sulfations, sulfonation,ethoxylation, propoxylation, amidation, and saponification. Themodification of the microalgal oil produces basic oleochemicals that canbe further modified into selected derivative oleochemicals for a desiredfunction. In a manner similar to that described above with reference tofuel producing processes, these chemical modifications can also beperformed on oils generated from the microbial cultures describedherein. Examples of basic oleochemicals include, but are not limited to,soaps, fatty acids, fatty esters, fatty alcohols, fatty nitrogencompounds, fatty acid methyl esters, and glycerol. Examples ofderivative oleochemicals include, but are not limited to, fattynitriles, esters, dimer acids, quats, surfactants, fatty alkanolamides,fatty alcohol sulfates, resins, emulsifiers, fatty alcohols, olefins,drilling muds, polyols, polyurethanes, polyacrylates, rubber, candles,cosmetics, metallic soaps, soaps, alpha-sulphonated methyl esters, fattyalcohol sulfates, fatty alcohol ethoxylates, fatty alcohol ethersulfates, imidazolines, surfactants, detergents, esters, quats,ozonolysis products, fatty amines, fatty alkanolamides, ethoxysulfates,monoglycerides, diglycerides, triglycerides (including medium chaintriglycerides), lubricants, hydraulic fluids, greases, dielectricfluids, mold release agents, metal working fluids, heat transfer fluids,other functional fluids, industrial chemicals (e.g., cleaners, textileprocessing aids, plasticizers, stabilizers, additives), surfacecoatings, paints and lacquers, electrical wiring insulation, and higheralkanes.

Hydrolysis of the fatty acid constituents from the glycerolipidsproduced by the methods of the invention yields free fatty acids thatcan be derivatized to produce other useful chemicals. Hydrolysis occursin the presence of water and a catalyst which may be either an acid or abase. The liberated free fatty acids can be derivatized to yield avariety of products, as reported in the following: U.S. Pat. No.5,304,664 (Highly sulfated fatty acids); U.S. Pat. No. 7,262,158(Cleansing compositions); U.S. Pat. No. 7,115,173 (Fabric softenercompositions); U.S. Pat. No. 6,342,208 (Emulsions for treating skin);U.S. Pat. No. 7,264,886 (Water repellant compositions); U.S. Pat. No.6,924,333 (Paint additives); U.S. Pat. No. 6,596,768 (Lipid-enrichedruminant feedstock); and U.S. Pat. No. 6,380,410 (Surfactants fordetergents and cleaners).

With regard to hydrolysis, in one embodiment of the invention, atriglyceride oil is optionally first hydrolyzed in a liquid medium suchas water or sodium hydroxide so as to obtain glycerol and soaps. Thereare various suitable triglyceride hydrolysis methods, including, but notlimited to, saponification, acid hydrolysis, alkaline hydrolysis,enzymatic hydrolysis (referred herein as splitting), and hydrolysisusing hot-compressed water. One skilled in the art will recognize that atriglyceride oil need not be hydrolyzed in order to produce anoleochemical; rather, the oil may be converted directly to the desiredoleochemical by other known process. For example, the triglyceride oilmay be directly converted to a methyl ester fatty acid throughesterification.

In some embodiments, catalytic hydrolysis of the oil produced by methodsdisclosed herein occurs by splitting the oil into glycerol and fattyacids. As discussed above, the fatty acids may then be further processedthrough several other modifications to obtained derivativeoleochemicals. For example, in one embodiment the fatty acids mayundergo an amination reaction to produce fatty nitrogen compounds. Inanother embodiment, the fatty acids may undergo ozonolysis to producemono- and dibasic-acids.

In other embodiments hydrolysis may occur via the, splitting of oilsproduced herein to create oleochemicals. In some preferred embodimentsof the invention, a triglyceride oil may be split before other processesis performed. One skilled in the art will recognize that there are manysuitable triglyceride splitting methods, including, but not limited to,enzymatic splitting and pressure splitting.

Generally, enzymatic oil splitting methods use enzymes, lipases, asbiocatalysts acting on a water/oil mixture. Enzymatic splitting thenslpits the oil or fat, respectively, is into glycerol and free fattyacids. The glycerol may then migrates into the water phase whereas theorganic phase enriches with free fatty acids.

The enzymatic splitting reactions generally take place at the phaseboundary between organic and aqueous phase, where the enzyme is presentonly at the phase boundary. Triglycerides that meet the phase boundarythen contribute to or participate in the splitting reaction. As thereaction proceeds, the occupation density or concentration of fattyacids still chemically bonded as glycerides, in comparison to free fattyacids, decreases at the phase boundary so that the reaction is sloweddown. In certain embodiments, enzymatic splitting may occur at roomtemperature. One of ordinary skill in the art would know the suitableconditions for splitting oil into the desired fatty acids.

By way of example, the reaction speed can be accelerated by increasingthe interface boundary surface. Once the reaction is complete, freefatty acids are then separated from the organic phase freed from enzyme,and the residue which still contains fatty acids chemically bonded asglycerides is fed back or recycled and mixed with fresh oil or fat to besubjected to splitting. In this manner, recycled glycerides are thensubjected to a further enzymatic splitting process. In some embodiments,the free fatty acids are extracted from an oil or fat partially split insuch a manner. In that way, if the chemically bound fatty acids(triglycerides) are returned or fed back into the splitting process, theenzyme consumption can be drastically reduced.

The splitting degree is determined as the ratio of the measured acidvalue divided by the theoretically possible acid value which can becomputed for a given oil or fat. Preferably, the acid value is measuredby means of titration according to standard common methods.Alternatively, the density of the aqueous glycerol phase can be taken asa measure for the splitting degree.

In one embodiment, the slitting process as described herein is alsosuitable for splitting the mono-, di- and triglyceride that arecontained in the so-called soap-stock from the alkali refining processesof the produced oils. In this manner, the soap-stock can bequantitatively converted without prior saponification of the neutraloils into the fatty acids. For this purpose, the fatty acids beingchemically bonded in the soaps are released, preferably beforesplitting, through an addition of acid. In certain embodiments, a buffersolution is used in addition to water and enzyme for the splittingprocess.

In one embodiment, oils produced in accordance with the methods of theinvention can also be subjected to saponification as a method ofhydrolysis Animal and plant oils are typically made of triacylglycerols(TAGs), which are esters of fatty acids with the trihydric alcohol,glycerol. In an alkaline hydrolysis reaction, the glycerol in a TAG isremoved, leaving three carboxylic acid anions that can associate withalkali metal cations such as sodium or potassium to produce fatty acidsalts. In this scheme, the carboxylic acid constituents are cleaved fromthe glycerol moiety and replaced with hydroxyl groups. The quantity ofbase (e.g., KOH) that is used in the reaction is determined by thedesired degree of saponification. If the objective is, for example, toproduce a soap product that comprises some of the oils originallypresent in the TAG composition, an amount of base insufficient toconvert all of the TAGs to fatty acid salts is introduced into thereaction mixture. Normally, this reaction is performed in an aqueoussolution and proceeds slowly, but may be expedited by the addition ofheat. Precipitation of the fatty acid salts can be facilitated byaddition of salts, such as water-soluble alkali metal halides (e.g.,NaCl or KCl), to the reaction mixture. Preferably, the base is an alkalimetal hydroxide, such as NaOH or KOH. Alternatively, other bases, suchas alkanolamines, including for example triethanolamine andaminomethylpropanol, can be used in the reaction scheme. In some cases,these alternatives may be preferred to produce a clear soap product. Inone embodiment the lipid composition subjected to saponification is atallow mimetic (i.e., lipid composition similar to that of tallow)produced as described herein, or a blend of a tallow mimetic withanother triglyceride oil.

In some methods, the first step of chemical modification may behydroprocessing to saturate double bonds, followed by deoxygenation atelevated temperature in the presence of hydrogen and a catalyst. Inother methods, hydrogenation and deoxygenation may occur in the samereaction. In still other methods deoxygenation occurs beforehydrogenation. Isomerization may then be optionally performed, also inthe presence of hydrogen and a catalyst. Finally, gases and naphthacomponents can be removed if desired. For example, see U.S. Pat. No.5,475,160 (hydrogenation of triglycerides); U.S. Pat. No. 5,091,116(deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815(hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).

In some embodiments of the invention, the triglyceride oils arepartially or completely deoxygenated. The deoxygenation reactions formdesired products, including, but not limited to, fatty acids, fattyalcohols, polyols, ketones, and aldehydes. In general, without beinglimited by any particular theory, the deoxygenation reactions involve acombination of various different reaction pathways, including withoutlimitation: hydrogenolysis, hydrogenation, consecutivehydrogenation-hydrogenolysis, consecutive hydrogenolysis-hydrogenation,and combined hydrogenation-hydrogenolysis reactions, resulting in atleast the partial removal of oxygen from the fatty acid or fatty acidester to produce reaction products, such as fatty alcohols, that can beeasily converted to the desired chemicals by further processing. Forexample, in one embodiment, a fatty alcohol may be converted to olefinsthrough FCC reaction or to higher alkanes through a condensationreaction.

One such chemical modification is hydrogenation, which is the additionof hydrogen to double bonds in the fatty acid constituents ofglycerolipids or of free fatty acids. The hydrogenation process permitsthe transformation of liquid oils into semi-solid or solid fats, whichmay be more suitable for specific applications.

Hydrogenation of oil produced by the methods described herein can beperformed in conjunction with one or more of the methods and/ormaterials provided herein, as reported in the following: U.S. Pat. No.7,288,278 (Food additives or medicaments); U.S. Pat. No. 5,346,724(Lubrication products); U.S. Pat. No. 5,475,160 (Fatty alcohols); U.S.Pat. No. 5,091,116 (Edible oils); U.S. Pat. No. 6,808,737 (Structuralfats for margarine and spreads); U.S. Pat. No. 5,298,637(Reduced-calorie fat substitutes); U.S. Pat. No. 6,391,815(Hydrogenation catalyst and sulfur adsorbent); U.S. Pat. Nos. 5,233,099and 5,233,100 (Fatty alcohols); U.S. Pat. No. 4,584,139 (Hydrogenationcatalysts); U.S. Pat. No. 6,057,375 (Foam suppressing agents); and U.S.Pat. No. 7,118,773 (Edible emulsion spreads).

One skilled in the art will recognize that various processes may be usedto hydrogenate carbohydrates. One suitable method includes contactingthe carbohydrate with hydrogen or hydrogen mixed with a suitable gas anda catalyst under conditions sufficient in a hydrogenation reactor toform a hydrogenated product. The hydrogenation catalyst generally caninclude Cu, Re, Ni, Fe, Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or anycombination thereof, either alone or with promoters such as W, Mo, Au,Ag, Cr, Zn, Mn, Sn, B, P, Bi, and alloys or any combination thereof.Other effective hydrogenation catalyst materials include eithersupported nickel or ruthenium modified with rhenium. In an embodiment,the hydrogenation catalyst also includes any one of the supports,depending on the desired functionality of the catalyst. Thehydrogenation catalysts may be prepared by methods known to those ofordinary skill in the art.

In some embodiments the hydrogenation catalyst includes a supportedGroup VIII metal catalyst and a metal sponge material (e.g., a spongenickel catalyst). Raney nickel provides an example of an activatedsponge nickel catalyst suitable for use in this invention. In otherembodiment, the hydrogenation reaction in the invention is performedusing a catalyst comprising a nickel-rhenium catalyst or atungsten-modified nickel catalyst. One example of a suitable catalystfor the hydrogenation reaction of the invention is a carbon-supportednickel-rhenium catalyst.

In an embodiment, a suitable Raney nickel catalyst may be prepared bytreating an alloy of approximately equal amounts by weight of nickel andaluminum with an aqueous alkali solution, e.g., containing about 25weight % of sodium hydroxide. The aluminum is selectively dissolved bythe aqueous alkali solution resulting in a sponge shaped materialcomprising mostly nickel with minor amounts of aluminum. The initialalloy includes promoter metals (i.e., molybdenum or chromium) in theamount such that about 1 to 2 weight % remains in the formed spongenickel catalyst. In another embodiment, the hydrogenation catalyst isprepared using a solution of ruthenium(III) nitrosylnitrate, ruthenium(III) chloride in water to impregnate a suitable support material. Thesolution is then dried to form a solid having a water content of lessthan about 1% by weight. The solid may then be reduced at atmosphericpressure in a hydrogen stream at 300° C. (uncalcined) or 400° C.(calcined) in a rotary ball furnace for 4 hours. After cooling andrendering the catalyst inert with nitrogen, 5% by volume of oxygen innitrogen is passed over the catalyst for 2 hours.

In certain embodiments, the catalyst described includes a catalystsupport. The catalyst support stabilizes and supports the catalyst. Thetype of catalyst support used depends on the chosen catalyst and thereaction conditions. Suitable supports for the invention include, butare not limited to, carbon, silica, silica-alumina, zirconia, titania,ceria, vanadia, nitride, boron nitride, heteropolyacids, hydroxyapatite,zinc oxide, chromia, zeolites, carbon nanotubes, carbon fullerene andany combination thereof.

The catalysts used in this invention can be prepared using conventionalmethods known to those in the art. Suitable methods may include, but arenot limited to, incipient wetting, evaporative impregnation, chemicalvapor deposition, wash-coating, magnetron sputtering techniques, and thelike.

The conditions for which to carry out the hydrogenation reaction willvary based on the type of starting material and the desired products.One of ordinary skill in the art, with the benefit of this disclosure,will recognize the appropriate reaction conditions. In general, thehydrogenation reaction is conducted at temperatures of 80° C. to 250°C., and preferably at 90° C. to 200° C., and most preferably at 100° C.to 150° C. In some embodiments, the hydrogenation reaction is conductedat pressures from 500 KPa to 14000 KPa.

The hydrogen used in the hydrogenolysis reaction of the currentinvention may include external hydrogen, recycled hydrogen, in situgenerated hydrogen, and any combination thereof. As used herein, theterm “external hydrogen” refers to hydrogen that does not originate fromthe biomass reaction itself, but rather is added to the system fromanother source.

In some embodiments of the invention, it is desirable to convert thestarting carbohydrate to a smaller molecule that will be more readilyconverted to desired higher hydrocarbons. One suitable method for thisconversion is through a hydrogenolysis reaction. Various processes areknown for performing hydrogenolysis of carbohydrates. One suitablemethod includes contacting a carbohydrate with hydrogen or hydrogenmixed with a suitable gas and a hydrogenolysis catalyst in ahydrogenolysis reactor under conditions sufficient to form a reactionproduct comprising smaller molecules or polyols. As used herein, theterm “smaller molecules or polyols” includes any molecule that has asmaller molecular weight, which can include a smaller number of carbonatoms or oxygen atoms than the starting carbohydrate. In an embodiment,the reaction products include smaller molecules that include polyols andalcohols. Someone of ordinary skill in the art would be able to choosethe appropriate method by which to carry out the hydrogenolysisreaction.

In some embodiments, a 5 and/or 6 carbon sugar or sugar alcohol may beconverted to propylene glycol, ethylene glycol, and glycerol using ahydrogenolysis catalyst. The hydrogenolysis catalyst may include Cr, Mo,W, Re, Mn, Cu, Cd, Fe, Co, Ni, Pt, Pd, Rh, Ru, Ir, Os, and alloys or anycombination thereof, either alone or with promoters such as Au, Ag, Cr,Zn, Mn, Sn, Bi, B, O, and alloys or any combination thereof. Thehydrogenolysis catalyst may also include a carbonaceous pyropolymercatalyst containing transition metals (e.g., chromium, molybdemum,tungsten, rhenium, manganese, copper, cadmium) or Group VIII metals(e.g., iron, cobalt, nickel, platinum, palladium, rhodium, ruthenium,iridium, and osmium). In certain embodiments, the hydrogenolysiscatalyst may include any of the above metals combined with an alkalineearth metal oxide or adhered to a catalytically active support. Incertain embodiments, the catalyst described in the hydrogenolysisreaction may include a catalyst support as described above for thehydrogenation reaction.

The conditions for which to carry out the hydrogenolysis reaction willvary based on the type of starting material and the desired products.One of ordinary skill in the art, with the benefit of this disclosure,will recognize the appropriate conditions to use to carry out thereaction. In general, they hydrogenolysis reaction is conducted attemperatures of 110° C. to 300° C., and preferably at 170° C. to 220°C., and most preferably at 200° C. to 225° C. In some embodiments, thehydrogenolysis reaction is conducted under basic conditions, preferablyat a pH of 8 to 13, and even more preferably at a pH of 10 to 12. Insome embodiments, the hydrogenolysis reaction is conducted at pressuresin a range between 60 KPa and 16500 KPa, and preferably in a rangebetween 1700 KPa and 14000 KPa, and even more preferably between 4800KPa and 11000 KPa.

The hydrogen used in the hydrogenolysis reaction of the currentinvention can include external hydrogen, recycled hydrogen, in situgenerated hydrogen, and any combination thereof.

In some embodiments, the reaction products discussed above may beconverted into higher hydrocarbons through a condensation reaction in acondensation reactor. In such embodiments, condensation of the reactionproducts occurs in the presence of a catalyst capable of forming higherhydrocarbons. While not intending to be limited by theory, it isbelieved that the production of higher hydrocarbons proceeds through astepwise addition reaction including the formation of carbon-carbon, orcarbon-oxygen bond. The resulting reaction products include any numberof compounds containing these moieties, as described in more detailbelow.

In certain embodiments, suitable condensation catalysts include an acidcatalyst, a base catalyst, or an acid/base catalyst. As used herein, theterm “acid/base catalyst” refers to a catalyst that has both an acid anda base functionality. In some embodiments the condensation catalyst caninclude, without limitation, zeolites, carbides, nitrides, zirconia,alumina, silica, aluminosilicates, phosphates, titanium oxides, zincoxides, vanadium oxides, lanthanum oxides, yttrium oxides, scandiumoxides, magnesium oxides, cerium oxides, barium oxides, calcium oxides,hydroxides, heteropolyacids, inorganic acids, acid modified resins, basemodified resins, and any combination thereof. In some embodiments, thecondensation catalyst can also include a modifier. Suitable modifiersinclude La, Y, Sc, P, B, Bi, Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, and anycombination thereof. In some embodiments, the condensation catalyst canalso include a metal. Suitable metals include Cu, Ag, Au, Pt, Ni, Fe,Co, Ru, Zn, Cd, Ga, In, Rh, Pd, Ir, Re, Mn, Cr, Mo, W, Sn, Os, alloys,and any combination thereof.

In certain embodiments, the catalyst described in the condensationreaction may include a catalyst support as described above for thehydrogenation reaction. In certain embodiments, the condensationcatalyst is self-supporting. As used herein, the term “self-supporting”means that the catalyst does not need another material to serve assupport. In other embodiments, the condensation catalyst in used inconjunction with a separate support suitable for suspending thecatalyst. In an embodiment, the condensation catalyst support is silica.

The conditions under which the condensation reaction occurs will varybased on the type of starting material and the desired products. One ofordinary skill in the art, with the benefit of this disclosure, willrecognize the appropriate conditions to use to carry out the reaction.In some embodiments, the condensation reaction is carried out at atemperature at which the thermodynamics for the proposed reaction arefavorable. The temperature for the condensation reaction will varydepending on the specific starting polyol or alcohol. In someembodiments, the temperature for the condensation reaction is in a rangefrom 80° C. to 500° C., and preferably from 125° C. to 450° C., and mostpreferably from 125° C. to 250° C. In some embodiments, the condensationreaction is conducted at pressures in a range between 0 Kpa to 9000 KPa,and preferably in a range between 0 KPa and 7000 KPa, and even morepreferably between 0 KPa and 5000 KPa.

The higher alkanes formed by the invention include, but are not limitedto, branched or straight chain alkanes that have from 4 to 30 carbonatoms, branched or straight chain alkenes that have from 4 to 30 carbonatoms, cycloalkanes that have from 5 to 30 carbon atoms, cycloalkenesthat have from 5 to 30 carbon atoms, aryls, fused aryls, alcohols, andketones. Suitable alkanes include, but are not limited to, butane,pentane, pentene, 2-methylbutane, hexane, hexene, 2-methylpentane,3-methylpentane, 2,2,-dimethylbutane, 2,3-dimethylbutane, heptane,heptene, octane, octene, 2,2,4-trimethylpentane, 2,3-dimethyl hexane,2,3,4-trimethylpentane, 2,3-dimethylpentane, nonane, nonene, decane,decene, undecane, undecene, dodecane, dodecene, tridecane, tridecene,tetradecane, tetradecene, pentadecane, pentadecene, nonyldecane,nonyldecene, eicosane, eicosene, uneicosane, uneicosene, doeicosane,doeicosene, trieicosane, trieicosene, tetraeicosane, tetraeicosene, andisomers thereof. Some of these products may be suitable for use asfuels.

In some embodiments, the cycloalkanes and the cycloalkenes areunsubstituted. In other embodiments, the cycloalkanes and cycloalkenesare mono-substituted. In still other embodiments, the cycloalkanes andcycloalkenes are multi-substituted. In the embodiments comprising thesubstituted cycloalkanes and cycloalkenes, the substituted groupincludes, without limitation, a branched or straight chain alkyl having1 to 12 carbon atoms, a branched or straight chain alkylene having 1 to12 carbon atoms, a phenyl, and any combination thereof. Suitablecycloalkanes and cycloalkenes include, but are not limited to,cyclopentane, cyclopentene, cyclohexane, cyclohexene,methyl-cyclopentane, methyl-cyclopentene, ethyl-cyclopentane,ethyl-cyclopentene, ethyl-cyclohexane, ethyl-cyclohexene, isomers andany combination thereof.

In some embodiments, the aryls formed are unsubstituted. In anotherembodiment, the aryls formed are mono-substituted. In the embodimentscomprising the substituted aryls, the substituted group includes,without limitation, a branched or straight chain alkyl having 1 to 12carbon atoms, a branched or straight chain alkylene having 1 to 12carbon atoms, a phenyl, and any combination thereof. Suitable aryls forthe invention include, but are not limited to, benzene, toluene, xylene,ethyl benzene, para xylene, meta xylene, and any combination thereof.

The alcohols produced in the invention have from 4 to 30 carbon atoms.In some embodiments, the alcohols are cyclic. In other embodiments, thealcohols are branched. In another embodiment, the alcohols are straightchained. Suitable alcohols for the invention include, but are notlimited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol,decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol,hexadecanol, heptyldecanol, octyldecanol, nonyldecanol, eicosanol,uneicosanol, doeicosanol, trieicosanol, tetraeicosanol, and isomersthereof.

The ketones produced in the invention have from 4 to 30 carbon atoms. Inan embodiment, the ketones are cyclic. In another embodiment, theketones are branched. In another embodiment, the ketones are straightchained. Suitable ketones for the invention include, but are not limitedto, butanone, pentanone, hexanone, heptanone, octanone, nonanone,decanone, undecanone, dodecanone, tridecanone, tetradecanone,pentadecanone, hexadecanone, heptyldecanone, octyldecanone,nonyldecanone, eicosanone, uneicosanone, doeicosanone, trieicosanone,tetraeicosanone, and isomers thereof.

Another such chemical modification is interesterification. Naturallyproduced glycerolipids do not have a uniform distribution of fatty acidconstituents. In the context of oils, interesterification refers to theexchange of acyl radicals between two esters of different glycerolipids.The interesterification process provides a mechanism by which the fattyacid constituents of a mixture of glycerolipids can be rearranged tomodify the distribution pattern. Interesterification is a well-knownchemical process, and generally comprises heating (to about 200° C.) amixture of oils for a period (e.g, 30 minutes) in the presence of acatalyst, such as an alkali metal or alkali metal alkylate (e.g., sodiummethoxide). This process can be used to randomize the distributionpattern of the fatty acid constituents of an oil mixture, or can bedirected to produce a desired distribution pattern. This method ofchemical modification of lipids can be performed on materials providedherein, such as microbial biomass with a percentage of dry cell weightas lipid at least 20%.

Directed interesterification, in which a specific distribution patternof fatty acids is sought, can be performed by maintaining the oilmixture at a temperature below the melting point of some TAGs whichmight occur. This results in selective crystallization of these TAGs,which effectively removes them from the reaction mixture as theycrystallize. The process can be continued until most of the fatty acidsin the oil have precipitated, for example. A directedinteresterification process can be used, for example, to produce aproduct with a lower calorie content via the substitution oflonger-chain fatty acids with shorter-chain counterparts. Directedinteresterification can also be used to produce a product with a mixtureof fats that can provide desired melting characteristics and structuralfeatures sought in food additives or products (e.g., margarine) withoutresorting to hydrogenation, which can produce unwanted trans isomers.

Interesterification of oils produced by the methods described herein canbe performed in conjuction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: U.S.Pat. No. 6,080,853 (Nondigestible fat substitutes); U.S. Pat. No.4,288,378 (Peanut butter stabilizer); U.S. Pat. No. 5,391,383 (Ediblespray oil); U.S. Pat. No. 6,022,577 (Edible fats for food products);U.S. Pat. No. 5,434,278 (Edible fats for food products); U.S. Pat. No.5,268,192 (Low calorie nut products); U.S. Pat. No. 5,258,197 (Reducecalorie edible compositions); U.S. Pat. No. 4,335,156 (Edible fatproduct); U.S. Pat. No. 7,288,278 (Food additives or medicaments); U.S.Pat. No. 7,115,760 (Fractionation process); U.S. Pat. No. 6,808,737(Structural fats); U.S. Pat. No. 5,888,947 (Engine lubricants); U.S.Pat. No. 5,686,131 (Edible oil mixtures); and U.S. Pat. No. 4,603,188(Curable urethane compositions).

In one embodiment in accordance with the invention, transesterificationof the oil, as described above, is followed by reaction of thetransesterified product with polyol, as reported in U.S. Pat. No.6,465,642, to produce polyol fatty acid polyesters. Such anesterification and separation process may comprise the steps as follows:reacting a lower alkyl ester with polyol in the presence of soap;removing residual soap from the product mixture; water-washing anddrying the product mixture to remove impurities; bleaching the productmixture for refinement; separating at least a portion of the unreactedlower alkyl ester from the polyol fatty acid polyester in the productmixture; and recycling the separated unreacted lower alkyl ester.

Transesterification can also be performed on microbial biomass withshort chain fatty acid esters, as reported in U.S. Pat. No. 6,278,006.In general, transesterification may be performed by adding a short chainfatty acid ester to an oil in the presence of a suitable catalyst andheating the mixture. In some embodiments, the oil comprises about 5% toabout 90% of the reaction mixture by weight. In some embodiments, theshort chain fatty acid esters can be about 10% to about 50% of thereaction mixture by weight. Non-limiting examples of catalysts includebase catalysts, sodium methoxide, acid catalysts including inorganicacids such as sulfuric acid and acidified clays, organic acids such asmethane sulfonic acid, benzenesulfonic acid, and toluenesulfonic acid,and acidic resins such as Amberlyst 15. Metals such as sodium andmagnesium, and metal hydrides also are useful catalysts.

Another such chemical modification is hydroxylation, which involves theaddition of water to a double bond resulting in saturation and theincorporation of a hydroxyl moiety. The hydroxylation process provides amechanism for converting one or more fatty acid constituents of aglycerolipid to a hydroxy fatty acid. Hydroxylation can be performed,for example, via the method reported in U.S. Pat. No. 5,576,027.Hydroxylated fatty acids, including castor oil and its derivatives, areuseful as components in several industrial applications, including foodadditives, surfactants, pigment wetting agents, defoaming agents, waterproofing additives, plasticizing agents, cosmetic emulsifying and/ordeodorant agents, as well as in electronics, pharmaceuticals, paints,inks, adhesives, and lubricants. One example of how the hydroxylation ofa glyceride may be performed is as follows: fat may be heated,preferably to about 30-50° C. combined with heptane and maintained attemperature for thirty minutes or more; acetic acid may then be added tothe mixture followed by an aqueous solution of sulfuric acid followed byan aqueous hydrogen peroxide solution which is added in small incrementsto the mixture over one hour; after the aqueous hydrogen peroxide, thetemperature may then be increased to at least about 60° C. and stirredfor at least six hours; after the stirring, the mixture is allowed tosettle and a lower aqueous layer formed by the reaction may be removedwhile the upper heptane layer formed by the reaction may be washed withhot water having a temperature of about 60° C.; the washed heptane layermay then be neutralized with an aqueous potassium hydroxide solution toa pH of about 5 to 7 and then removed by distillation under vacuum; thereaction product may then be dried under vacuum at 100° C. and the driedproduct steam-deodorized under vacuum conditions and filtered at about50° to 60° C. using diatomaceous earth.

Hydroxylation of microbial oils produced by the methods described hereincan be performed in conjuction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: U.S.Pat. No. 6,590,113 (Oil-based coatings and ink); U.S. Pat. No. 4,049,724(Hydroxylation process); U.S. Pat. No. 6,113,971 (Olive oil butter);U.S. Pat. No. 4,992,189 (Lubricants and lube additives); U.S. Pat. No.5,576,027 (Hydroxylated milk); and U.S. Pat. No. 6,869,597 (Cosmetics).

Hydroxylated glycerolipids can be converted to estolides. Estolidesconsist of a glycerolipid in which a hydroxylated fatty acid constituenthas been esterified to another fatty acid molecule. Conversion ofhydroxylated glycerolipids to estolides can be carried out by warming amixture of glycerolipids and fatty acids and contacting the mixture witha mineral acid, as described by Isbell et al., JAOCS 71(2):169-174(1994). Estolides are useful in a variety of applications, includingwithout limitation those reported in the following: U.S. Pat. No.7,196,124 (Elastomeric materials and floor coverings); U.S. Pat. No.5,458,795 (Thickened oils for high-temperature applications); U.S. Pat.No. 5,451,332 (Fluids for industrial applications); U.S. Pat. No.5,427,704 (Fuel additives); and U.S. Pat. No. 5,380,894 (Lubricants,greases, plasticizers, and printing inks).

Another such chemical modification is olefin metathesis. In olefinmetathesis, a catalyst severs the alkylidene carbons in an alkene(olefin) and forms new alkenes by pairing each of them with differentalkylidine carbons. The olefin metathesis reaction provides a mechanismfor processes such as truncating unsaturated fatty acid alkyl chains atalkenes by ethenolysis, cross-linking fatty acids through alkenelinkages by self-metathesis, and incorporating new functional groups onfatty acids by cross-metathesis with derivatized alkenes.

In conjunction with other reactions, such as transesterification andhydrogenation, olefin metathesis can transform unsaturated glycerolipidsinto diverse end products. These products include glycerolipid oligomersfor waxes; short-chain glycerolipids for lubricants; homo- andhetero-bifunctional alkyl chains for chemicals and polymers; short-chainesters for biofuel; and short-chain hydrocarbons for jet fuel. Olefinmetathesis can be performed on triacylglycerols and fatty acidderivatives, for example, using the catalysts and methods reported inU.S. Pat. No. 7,119,216, US Patent Pub. No. 2010/0160506, and U.S.Patent Pub. No. 2010/0145086.

Olefin metathesis of bio-oils generally comprises adding a solution ofRu catalyst at a loading of about 10 to 250 ppm under inert conditionsto unsaturated fatty acid esters in the presence (cross-metathesis) orabsence (self-metathesis) of other alkenes. The reactions are typicallyallowed to proceed from hours to days and ultimately yield adistribution of alkene products. One example of how olefin metathesismay be performed on a fatty acid derivative is as follows: A solution ofthe first generation Grubbs Catalyst(dichloro[2(1-methylethoxy-α-O)phenyl]methylene-α-C](tricyclohexyl-phosphine) in toluene at a catalyst loading of 222 ppmmay be added to a vessel containing degassed and dried methyl oleate.Then the vessel may be pressurized with about 60 psig of ethylene gasand maintained at or below about 30° C. for 3 hours, wherebyapproximately a 50% yield of methyl 9-decenoate may be produced.

Olefin metathesis of oils produced by the methods described herein canbe performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: PatentApp. PCT/US07/081,427 (α-olefin fatty acids) and U.S. patent applicationSer. No. 12/281,938 (petroleum creams), Ser. No. 12/281,931 (paintballgun capsules), Ser. No. 12/653,742 (plasticizers and lubricants), Ser.No. 12/422,096 (bifunctional organic compounds), and Ser. No. 11/795,052(candle wax).

Other chemical reactions that can be performed on microbial oils includereacting triacylglycerols with a cyclopropanating agent to enhancefluidity and/or oxidative stability, as reported in U.S. Pat. No.6,051,539; manufacturing of waxes from triacylglycerols, as reported inU.S. Pat. No. 6,770,104; and epoxidation of triacylglycerols, asreported in “The effect of fatty acid composition on the acrylationkinetics of epoxidized triacylglycerols”, Journal of the American OilChemists' Society, 79:1, 59-63, (2001) and Free Radical Biology andMedicine, 37:1, 104-114 (2004).

The generation of oil-bearing microbial biomass for fuel and chemicalproducts as described above results in the production of delipidatedbiomass meal. Delipidated meal is a byproduct of preparing algal oil andis useful as animal feed for farm animals, e.g., ruminants, poultry,swine and aquaculture. The resulting meal, although of reduced oilcontent, still contains high quality proteins, carbohydrates, fiber,ash, residual oil and other nutrients appropriate for an animal feed.Because the cells are predominantly lysed by the oil separation process,the delipidated meal is easily digestible by such animals. Delipidatedmeal can optionally be combined with other ingredients, such as grain,in an animal feed. Because delipidated meal has a powdery consistency,it can be pressed into pellets using an extruder or expander or anothertype of machine, which are commercially available.

The invention, having been described in detail above, is exemplified inthe following examples, which are offered to illustrate, but not tolimit, the claimed invention.

VII. Examples Example 1 Methods for Culturing Prototheca

Prototheca strains were cultivated to achieve a high percentage of oilby dry cell weight. Cryopreserved cells were thawed at room temperatureand 500 ul of cells were added to 4.5 ml of medium (4.2 g/L K₂HPO₄, 3.1g/L NaH₂PO₄, 0.24 g/L MgSO₄.7H₂O, 0.25 g/L Citric Acid monohydrate,0.025 g/L CaCl₂ 2H₂O, 2 g/L yeast extract) plus 2% glucose and grown for7 days at 28° C. with agitation (200 rpm) in a 6-well plate. Dry cellweights were determined by centrifuging 1 ml of culture at 14,000 rpmfor 5 mM in a pre-weighed Eppendorf tube. The culture supernatant wasdiscarded and the resulting cell pellet washed with 1 ml of deionizedwater. The culture was again centrifuged, the supernatant discarded, andthe cell pellets placed at −80° C. until frozen. Samples were thenlyophilized for 24 hrs and dry cell weights calculated. Fordetermination of total lipid in cultures, 3 ml of culture was removedand subjected to analysis using an Ankom system (Ankom Inc., Macedon,N.Y.) according to the manufacturer's protocol. Samples were subjectedto solvent extraction with an Amkom XT10 extractor according to themanufacturer's protocol. Total lipid was determined as the difference inmass between acid hydrolyzed dried samples and solvent extracted, driedsamples. Percent oil dry cell weight measurements are shown in Table 10.

TABLE 10 Percent oil by dry cell weight Species Strain % Oil Protothecastagnora UTEX 327 13.14 Prototheca moriformis UTEX 1441 18.02 Protothecamoriformis UTEX 1435 27.17

Microalgae samples from multiple strains from the genus Prototheca weregenotyped. Genomic DNA was isolated from algal biomass as follows. Cells(approximately 200 mg) were centifuged from liquid cultures 5 minutes at14,000×g. Cells were then resuspended in sterile distilled water,centrifuged 5 minutes at 14,000×g and the supernatant discarded. Asingle glass bead ˜2 mm in diameter was added to the biomass and tubeswere placed at −80° C. for at least 15 minutes. Samples were removed and150 μl of grinding buffer (1% Sarkosyl, 0.25 M Sucrose, 50 mM NaCl, 20mM EDTA, 100 mM Tris-HCl, pH 8.0, RNase A 0.5 ug/ul) was added. Pelletswere resuspended by vortexing briefly, followed by the addition of 40 ulof 5M NaCl. Samples were vortexed briefly, followed by the addition of66 μl of 5% CTAB (Cetyl trimethylammonium bromide) and a final briefvortex. Samples were next incubated at 65° C. for 10 minutes after whichthey were centrifuged at 14,000×g for 10 minutes. The supernatant wastransferred to a fresh tube and extracted once with 300 μl ofPhenol:Chloroform:Isoamyl alcohol 12:12:1, followed by centrifugationfor 5 minutes at 14,000×g. The resulting aqueous phase was transferredto a fresh tube containing 0.7 vol of isopropanol (˜190 μl), mixed byinversion and incubated at room temperature for 30 minutes or overnightat 4° C. DNA was recovered via centrifugation at 14,000×g for 10minutes. The resulting pellet was then washed twice with 70% ethanol,followed by a final wash with 100% ethanol. Pellets were air dried for20-30 minutes at room temperature followed by resuspension in 50 μl of10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μl of total algal DNA, prepared as described above, was diluted1:50 in 10 mM Tris, pH 8.0. PCR reactions, final volume 20 μl, were setup as follows. Ten μl of 2× iProof HF master mix (BIO-RAD) was added to0.4 μl primer SZ02613 (5′-TGTTGAAGAATGAGCCGGCGAC-3′ (SEQ ID NO:9) at 10mM stock concentration). This primer sequence runs from position 567-588in Gen Bank accession no. L43357 and is highly conserved in higherplants and algal plastid genomes. This was followed by the addition of0.4 μl primer SZ02615 (5′-CAGTGAGCTATTACGCACTC-3′ (SEQ ID NO:10) at 10mM stock concentration). This primer sequence is complementary toposition 1112-1093 in Gen Bank accession no. L43357 and is highlyconserved in higher plants and algal plastid genomes. Next, 5 μl ofdiluted total DNA and 3.2 μl dH₂O were added. PCR reactions were run asfollows: 98° C., 45″; 98° C., 8″; 53° C., 12″; 72° C., 20″ for 35 cyclesfollowed by 72° C. for 1 min and holding at 25° C. For purification ofPCR products, 20 μl of 10 mM Tris, pH 8.0, was added to each reaction,followed by extraction with 40 μl of Phenol:Chloroform:isoamyl alcohol12:12:1, vortexing and centrifuging at 14,000×g for 5 minutes. PCRreactions were applied to S-400 columns (GE Healthcare) and centrifugedfor 2 minutes at 3,000×g. Purified PCR products were subsequently TOPOcloned into PCR8/GW/TOPO and positive clones selected for on LB/Specplates. Purified plasmid DNA was sequenced in both directions using M13forward and reverse primers. In total, twelve Prototheca strains wereselected to have their 23S rRNA DNA sequenced and the sequences arelisted in the Sequence Listing. A summary of the strains and SequenceListing Numbers is included below. The sequences were analyzed foroverall divergence from the UTEX 1435 (SEQ ID NO: 15) sequence. Twopairs emerged (UTEX 329/UTEX 1533 and UTEX 329/UTEX 1440) as the mostdivergent. In both cases, pairwise alignment resulted in 75.0% pairwisesequence identity. The percent sequence identity to UTEX 1435 is alsoincluded below:

% Species Strain nt identity SEQ ID NO. Prototheca kruegani UTEX 32975.2 SEQ ID NO: 11 Prototheca wickerhamii UTEX 1440 99 SEQ ID NO: 12Prototheca stagnora UTEX 1442 75.7 SEQ ID NO: 13 Prototheca moriformisUTEX 288 75.4 SEQ ID NO: 14 Prototheca moriformis UTEX 1439; 100 SEQ IDNO: 15 1441; 1435; 1437 Prototheca wikerhamii UTEX 1533 99.8 SEQ ID NO:16 Prototheca moriformis UTEX 1434 75.9 SEQ ID NO: 17 Prototheca zopfiiUTEX 1438 75.7 SEQ ID NO: 18 Prototheca moriformis UTEX 1436 88.9 SEQ IDNO: 19

Lipid samples from a subset of the above-listed strains were analyzedfor lipid profile using HPLC. Results are shown below in Table 11.

TABLE 11 Diversity of lipid chains in Prototheca species Strain C14:0C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 UTEX 0 12.01 0 0 50.3317.14 0 0 0 327 UTEX 1.41 29.44 0.70 3.05 57.72 12.37 0.97 0.33 0 1441UTEX 1.09 25.77 0 2.75 54.01 11.90 2.44 0 0 1435

Oil extracted from Prototheca moriformis UTEX 1435 (via solventextraction or using an expeller press was analyzed for carotenoids,chlorophyll, tocopherols, other sterols and tocotrienols. The resultsare summarized below in Table 12.

TABLE 12 Carotenoid, chlorophyll, tocopherol/sterols and tocotrienolanalysis in oil extracted from Prototheca moriformis (UTEX 1435).Pressed oil Solvent extracted (mcg/ml) oil (mcg/ml) cis-Lutein 0.0410.042 trans-Lutein 0.140 0.112 trans-Zeaxanthin 0.045 0.039cis-Zeaxanthin 0.007 0.013 t-alpha-Crytoxanthin 0.007 0.010t-beta-Crytoxanthin 0.009 0.010 t-alpha-Carotene 0.003 0.001c-alpha-Carotene none detected none detected t-beta-Carotene 0.010 0.0099-cis-beta-Carotene 0.004 0.002 Lycopene none detected none detectedTotal Carotenoids 0.267 0.238 Chlorophyll <0.01 mg/kg <0.01 mg/kgTocopherols and Sterols Pressed oil Solvent extracted (mg/100 g) oil(mg/100 g) gamma Tocopherol 0.49 0.49 Campesterol 6.09 6.05 Stigmasterol47.6 47.8 Beta-sitosterol 11.6 11.5 Other sterols 445 446 TocotrienolsPressed oil Solvent extracted (mg/g) oil (mg/g) alpha Tocotrienol 0.260.26 beta Tocotrienol <0.01 <0.01 gamma Tocotrienol 0.10 0.10 detalTocotrienol <0.01 <0.01 Total Tocotrienols 0.36 0.36

Oil extracted from Prototheca moriformis, from four separate lots, wererefined and bleached using standard vegetable oil processing methods.Briefly, crude oil extracted from Prototheca moriformis was clarified ina horizontal decanter, where the solids were separated from the oil. Theclarified oil was then transferred to a tank with citric acid and waterand left to settle for approximately 24 hours. After 24 hours, themixture in the tank formed 2 separate layers. The bottom layer wascomposed of water and gums that were then removed by decantation priorto transferring the degummed oil into a bleaching tank. The oil was thenheated along with another dose of citric acid. Bleaching clay was thenadded to the bleaching tank and the mixture was further heated undervacuum in order to evaporate off any water that was present. The mixturewas then pumped through a leaf filter in order to remove the bleachingclay. The filtered oil was then passed through a final 5 μm polishingfilter and then collected for storage until use. The refined andbleached (RB) oil was then analyzed for carotenoids, chlorophyll,sterols, tocotrienols and tocopherols. The results of these analyses aresummarized in Table 13 below. “Nd” denotes none detected and thesensitivity of detection is listed below:

Sensitivity of Detection

Carotenoids (mcg/g) nd=<0.003 mcg/g

Chlorophyll (mcg/g) nd=<0.03 mcg/g

Sterols (%) nd=0.25%

Tocopherols (mcg/g); nd=3 mcg/g

TABLE 13 Carotenoid, chlorophyll, sterols, tocotrienols and tocopherolanalysis from refined and bleached Prototheca moriformis oil. Lot A LotB Lot C Lot D Carotenoids (mcg/g) Lutein 0.025 0.003 nd 0.039 Zeaxanthinnd nd nd nd cis-Lutein/Zeaxanthin nd nd nd nd trans-alpha-Cryptoxanthinnd nd nd nd trans-beta-Cryptoxanthin nd nd nd nd trans-alpha-Carotene ndnd nd nd cis-alpha-Carotene nd nd nd nd trans-beta-Carotene nd nd nd ndcis-beta-Carotene nd nd nd nd Lycopene nd nd nd nd Unidentified 0.2190.066 0.050 0.026 Total Carotenoids 0.244 0.069 0.050 0.065 Chlorophyll(mcg/g) Chlorophyll A 0.268 0.136 0.045 0.166 Chlorophyll B nd nd nd ndTotal Chlorophyll 0.268 0.136 0.045 0.166 Sterols (%) Brassicasterol ndnd nd nd Campesterol nd nd nd nd Stigmasterol nd nd nd ndbeta-Sitosterol nd nd nd nd Total Sterols nd nd nd nd Tocopherols(mcg/g) alpha-Tocopherol 23.9 22.8 12.5 8.2 beta-Tocopherol 3.72 nd ndnd gamma-Tocopherol 164 85.3 43.1 38.3 delta-Tocopherol 70.1 31.1 18.114.3 Total Tocopherols 262 139.2 73.7 60.8 Tocotrienols (mcg/g)alpha-Tocotrienol 190 225 253 239 beta-Tocotrienol nd nd nd ndgamma-Tocotrienol 47.3 60.4 54.8 60.9 delta-Tocotrienol 12.3 16.1 17.515.2 Total Tocotrienols 250 302 325 315

The same four lots of Prototheca moriformis oil was also analyzed fortrace elements and the results are summarized below in Table 14.

TABLE 14 Elemental analysis of refined and bleached Protothecamoriformis oil. Lot A Lot B Lot C Lot D Elemental Analysis (ppm) Calcium0.08 0.07 <0.04 0.07 Phosphorous <0.2 0.38 <0.2 0.33 Sodium <0.5 0.55<0.5 <0.5 Potassium 1.02 1.68 <0.5 0.94 Magnesium <0.04 <0.04 <0.04 0.07Manganese <0.05 <0.05 <0.05 <0.05 Iron <0.02 <0.02 <0.02 <0.02 Zinc<0.02 <0.02 <0.02 <0.02 Copper <0.05 <0.05 <0.05 <0.05 Sulfur 2.55 4.452.36 4.55 Lead <0.2 <0.2 <0.2 <0.2 Silicon 0.37 0.41 0.26 0.26 Nickel<0.2 <0.2 <0.2 <0.2 Organic chloride <1.0 <1.0 <1.0 2.2 Inorganicchloride <1.0 <1.0 <1.0 <1.0 Nitrogen 4.4 7.8 4.2 6.9 Lithium <0.02<0.02 <0.02 <0.02 Boron 0.07 0.36 0.09 0.38 Aluminum — <0.2 <0.2 <0.2Vanadium <0.05 <0.05 <0.05 <0.05 Lovibond Color (°L) Red 5.0 4.3 3.2 5.0Yellow 70.0 70.0 50.0 70.0 Mono & Diglycerides by HPLC (%) Diglycerides1.68 2.23 1.25 1.61 Monoglycerides 0.03 0.04 0.02 0.03 Free fatty acids(FFA) 1.02 1.72 0.86 0.83 Soaps 0 0 0 Oxidized and PolymerizedTriglycerides Oxidized Triglycerides (%) 3.41 2.41 4.11 1.00 PolymerizedTriglycerides 1.19 0.45 0.66 0.31 (%) Peroxide Value (meg/kg) 0.75 0.800.60 1.20 p-Anisidine value 5.03 9.03 5.44 20.1 (dimensionless) Waterand Other Impurities (%) Karl Fisher Moisture 0.8 0.12 0.07 0.18 Totalpolar compounds 5.02 6.28 4.54 5.23 Unsaponificable matter 0.92 1.070.72 1.04 Insoluble impurities <0.01 <0.01 0.01 <0.01 Total oil (%)Neutral oil 98.8 98.2 99.0 98.9

Example 2 General Methods for Biolistic Transforation of Prototheca

Seashell Gold Microcarriers 550 nanometers were prepared according tothe protocol from manufacturer. Plasmid (20 μg) was mixed with 50 μl ofbinding buffer and 60 μl (30 mg) of S550d gold carriers and incubated inice for 1 min. Precipitation buffer (100 μl) was added, and the mixturewas incubated in ice for another 1 min. After vortexing, DNA-coatedparticles were pelleted by spinning at 10,000 rpm in an Eppendorf 5415Cmicrofuge for 10 seconds. The gold pellet was washed once with 500 μl ofcold 100% ethanol, pelleted by brief spinning in the microfuge, andresuspended with 50 μl of ice-cold ethanol. After a brief (1-2 sec)sonication, 10 μl of DNA-coated particles were immediately transferredto the carrier membrane.

Prototheca strains were grown in proteose medium (2 g/L yeast extract,2.94 mM NaNO3, 0.17 mM CaCl2.2H2O, 0.3 mM MgSO4.7H₂O, 0.4 mM K2HPO4,1.28 mM KH2PO4, 0.43 mM NaCl) with 2% glucose on a gyratory shaker untilit reaches a cell density of 2×10⁶ cells/ml. The cells were harvested,washed once with sterile distilled water, and resuspended in 50 μl ofmedium. 1×10⁷ cells were spread in the center third of a non-selectiveproteose media plate. The cells were bombarded with the PDS-1000/HeBiolistic Particle Delivery system (Bio-Rad). Rupture disks (1350 psi)were used, and the plates are placed 6 cm below the screen/macrocarrierassembly. The cells were allowed to recover at 25° C. for 12-24 h. Uponrecovery, the cells were scraped from the plates with a rubber spatula,mixed with 100 μl of medium and spread on plates containing theappropriate antibiotic selection. After 7-10 days of incubation at 25°C., colonies representing transformed cells were visible on the plates.Colonies were picked and spotted on selective (either antibiotic orcarbon source) agar plates for a second round of selection.

Example 3 Transformation of Chlorella

Vector Construction

A BamHI-SacII fragment containing the CMV promoter, a hygromycinresistance cDNA, and a CMV 3′ UTR (SEQ ID NO: 152, a subsequence of thepCAMBIA1380 vector, Cambia, Can berra, Australia) was cloned into theBamHI and SacII sites of pBluescript and is referred to herein as pHyg.

Biolistic Transformation of Chlorella

S550d gold carriers from Seashell Technology were prepared according tothe protocol from manufacturer. Linearized pHyg plasmid (20 μg) wasmixed with 50 μl of binding buffer and 60 μl (30 mg) of S550d goldcarriers and incubated in ice for 1 min. Precipitation buffer (100 μl)was added, and the mixture was incubated in ice for another 1 min. Aftervortexing, DNA-coated particles were pelleted by spinning at 10,000 rpmin an Eppendorf 5415C microfuge for 10 seconds. The gold pellet waswashed once with 500 μl of cold 100% ethanol, pelleted by brief spinningin the microfuge, and resuspended with 50 μl of ice-cold ethanol. Aftera brief (1-2 sec) sonication, 10 μl of DNA-coated particles wereimmediately transferred to the carrier membrane.

Chlorella protothecoides culture (Univeristy of Texas Culture Collection250) was grown in proteose medium (2 g/L yeast extract, 2.94 mM NaNO3,0.17 mM CaCl2.2H2O, 0.3 mM MgSO4.7H2O, 0.4 mM K2HPO4, 1.28 mM KH2PO4,0.43 mM NaCl) on a gyratory shaker under continuous light at 75 mmolphotons m⁻² sec⁻¹ until it reached a cell density of 2×10⁶ cells/ml. Thecells were harvested, washed once with sterile distilled water, andresuspended in 50 μl of medium. 1×10⁷ cells were spread in the centerthird of a non-selective proteose media plate. The cells were bombardedwith the PDS-1000/He Biolistic Particle Delivery system (Bio-Rad).Rupture disks (1100 and 1350 psi) were used, and the plates were placed9 and 12 cm below the screen/macrocarrier assembly. The cells wereallowed to recover at 25° C. for 12-24 h. Upon recovery, the cells werescraped from the plates with a rubber spatula, mixed with 100 μl ofmedium and spread on hygromycin contained plates (200 μg/ml). After 7-10days of incubation at 25° C., colonies representing transformed cellswere visible on the plates from 1100 and 1350 psi rupture discs and from9 and 12 cm distances. Colonies were picked and spotted on selectiveagar plates for a second round of selection.

Transformation of Chlorella by Electroporation

Chlorella protothecoides culture was grown in proteose medium on agyratory shaker under continuous light at 75 μmol photons m⁻² sec⁻¹until it reached a cell density of 2×10⁶ cells/ml. The cells wereharvested, washed once with sterile distilled water, and resuspended ina tris-phosphate buffer (20 m M Tris-HCl, pH 7.0; 1 mM potassiumphosphate) containing 50 mM sucrose to a density of 4×10⁸ cells/ml.About 250 μl cell suspension (1×10⁸ cells) was placed in a disposableelectroporation cuvette of 4 mm gap. To the cell suspension, 5 μg oflinearized pHyg plasmid DNA and 200 μg of carrier DNA (sheared salmonsperm DNA) was added. The electroporation cuvette was then incubated ina water bath at 16° C. for 10 minutes. An electrical pulse (1100 V/cm)was then applied to the cuvette at a capacitance of 25 μF (no shuntresistor was used for the electroporation) using a Gene Pulser II(Bio-Rad Labs, Hercules, Calif.) electroporation apparatus. The cuvettewas then incubated at room temperature for 5 minutes, following whichthe cell suspension was transferred to 50 ml of proteose media, andshaken on a gyratory shaker for 2 days. Following recovery, the cellswere harvested by centrifugation at low speed, resuspended in proteosemedia, and plated at low density on plates supplemented with 200 μg/mlhygromycin. The plates were incubated under continuous light at 75 μmolphotons m⁻² sec⁻¹. Transformants appeared as colonies in 1-2 weeks.Colonies were picked and spotted on selective agar plates for a secondround of selection.

Genotyping

A subset of colonies that survived a second round of selection werecultured in small volume and harvested. Pellets of approximately 5-10 uLvolume were resuspended in 50 uL of 10 mM NaEDTA by vortexing and thenincubated at 100° C. for 10. The tubes were then vortexed briefly andsonicated for 10 seconds, then centifuged at 12,000×g for 1 minute. 2 uLof supernatant as template was used in a 50 uL PCR reaction. Primersused for genotyping were SEQ ID NO: 153 and SEQ ID NO: 154. PCRconditions were as follows: 95° C. 5 mM×1 cycle; 95° C. 30 sec-58° C. 30sec-72° C. 1 mM 30 sec×35 cycles; 72° C. 10 min×1 cycle. The expected992 bp fragment was found in 6 of 10 colonies from the biolistic methodand from a single electroporation colony. A lower sized, nonspecificband was present in all lanes. To confirm the identity of the amplified992 bp fragment, two biolistic bands and the electroporation band wereexcised from the gel and individually sequenced. The sequence of allthree bands corresponded to the expected 992 bp fragment. (DNA ladder:Bionexus® All Purpose Hi-Lo® DNA ladder catalog #BN2050).

Example 4 Algal-derived Promoters and Genes for Use in Microalgae

A. 5′UTR and Promoter Sequences from Chlorella protothecoides

A cDNA library was generated from mixotrophically grown Chlorellaprotothecoides (UTEX 250) using standard techniques. Based upon the cDNAsequences, primers were designed in certain known housekeeping genes to“walk” upstream of the coding regions using Seegene's DNA Walking kit(Rockville, Md.). Sequences isolated include an actin (SEQ ID NO: 155)and elongation factor-1a (EF1a) (SEQ ID NO: 156) promoter/UTR, both ofwhich contain introns (as shown in the lower case) and exons (upper caseitalicized) and the predicted start site (in bold) and two beta-tubulinpromoter/UTR elements: Isoform A (SEQ ID NO: 157) and Isoform B (SEQ IDNO: 158).

B. Lipid Biosynthesis Enzyme and Plastid Targeting Sequences from C.protothecoides

From the cDNA library described above, three cDNAs encoding proteinsfunctional in lipid metabolism in Chlorella protothecoides (UTEX 250)were cloned using the same methods as described above. The nucleotideand amino acid sequences for an acyl ACP desaturase (SEQ ID NOs: 159 and160) and two geranyl geranyl diphosphate synthases (SEQ ID NOs: 161-164)are included in the Sequence Listing below. Additionally, three cDNAswith putative signal sequences targeting to the plastid were alsocloned. The nucleotide and amino acid sequences for aglyceraldehyde-3-phosphate dehydrogenase (SEQ ID NOs: 165 and 166), anoxygen evolving complex protein OEE33 (SEQ ID NOs: 167 and 168) and aClp protease (SEQ ID NOs: 169 and 170) are included in the SequenceListing below. The putative plastid targeting sequence has beenunderlined in both the nucleotide and amino acid sequence. The plastidtargeting sequences can be used to target the producs of transgenes tothe plastid of microbes, such as lipid modification enzymes.

Example 5 Genetic Engineering of Chlorella protothecoides to Express anExogenous Sucrose Invertase

Strains and Media:

Chlorella protothecoides (UTEX 250) was obtained from the CultureCollection of Alga at the University of Texas (Austin, Tex., USA). Thestock cultures were maintained on modified Proteose medium. ModifiedProteose medium consists of 0.25 g NaNO₃, 0.09 g K₂HPO₄, 0.175 g KH₂PO₄0.025 g, 0.025 g CaCl₂.2H₂O, 0.075 g MgSO₄.7H₂O, and 2 g yeast extractper liter (g/L).

Plasmid Construction:

To express the secreted form of invertase in Chlorella protothecoides, aSaccharomyces cerevisiae SUC2 gene was placed under the control of threedifferent promoters: Cauliflower mosaic virus ³⁵S promoter (CMV),Chlorella virus promoter (NC-1A), and Chlorella HUP1 promoter. A yeastSUC2 gene was synthesized to accommodate codon usage optimized for C.protothecoides and includes a signal sequence required for directingextracellular secretion of invertase. Each construct was built inpBluescript KS+, and EcoRI/AscI, AscI/XhoI, and XhoI/BamHI sites wereintroduced to each promoter, invertase gene, and CMV 3′UTR,respectively, by PCR ampilication using specific primers. Purified PCRproducts were cloned sequentially.

Transformation of Chlorella protothecoides:

A Chlorella protothecoides culture was grown in modified Proteose mediumon a gyratory shaker under continuous light at 75 μmol photons m⁻² sec⁻¹till it reached a cell density of 6×10⁶ cells/ml.

For biolistic transformation, S550d gold carriers from SeashellTechnology were prepared according to the protocol from themanufacturer. Briefly, a linearized construct (20 μg) by BsaI was mixedwith 50 μl of binding buffer and 60 μl (3 mg) of S550d gold carriers andincubated in ice for 1 min. Precipitation buffer (100 μl) was added, andthe mixture was incubated in ice for another 1 min. After mildvortexing, DNA-coated particles were pelleted by spinning at 10,000 rpmin an Eppendorf microfuge for 10 seconds. The gold pellet was washedonce with 500 μl of cold 100% ethanol, pelleted by brief spinning in themicrofuge, and resuspended with 50 μl of ice-cold ethanol. After a brief(1-2 sec) sonication, 10 μl of DNA-coated particles were immediatelytransferred to the carrier membrane. The cells were harvested, washedonce with sterile distilled water, resuspended in 50 μl of medium (1×10⁷cells), and were spread in the center third of a non-selective Proteousplate. The cells were bombarded with the PDS-1000/He Biolistic ParticleDelivery system (Bio-Rad). Rupture disks (1100 and 1350 psi) were used,and the plates were placed 9-12 cm below the screen/macrocarrierassembly. The cells were allowed to recover at 25° C. for 12-24 hours.Upon recovery, the cells were scraped from the plates with a rubberspatula, mixed with 100 μl of medium and spread on modified Proteoseplates with 1% sucrose. After 7-10 days of incubation at 25° C. in thedark, colonies representing transformed cells were visible on theplates.

For transformation with electroporation, cells were harvested, washedonce with sterile distilled water, and resuspended in a Tris-phosphatebuffer (20 m M Tris-HCl, pH 7.0; 1 mM potassium phosphate) containing 50mM sucrose to a density of 4×10⁸ cells/ml. About 250 μl cell suspension(1×10⁸ cells) was placed in a disposable electroporation cuvette of 4 mmgap. To the cell suspension, 5 μg of linearized plasmid DNA and 200 μgof carrier DNA (sheared salmon sperm DNA) were added. Theelectroporation cuvette was then incubated in an ice water bath at 16°C. for 10 mM. An electrical pulse (1100 V/cm) was then applied to thecuvette at a capacitance of 25 μF (no shunt resistor was used for theelectroporation) using a Gene Pulser II (Bio-Rad Labs, Hercules, Calif.)electroporation apparatus. The cuvette was then incubated at roomtemperature for 5 minutes, following which the cell suspension wastransferred to 50 ml of modified Proteose media, and shaken on agyratory shaker for 2 days. Following recovery, the cells were harvestedat low speed (4000 rpm), resuspended in modified Proteose media, andplated out at low density on modified Proteose plates with 1% sucrose.After 7-10 days of incubation at 25° C. in the dark, coloniesrepresenting transformed cells were visible on the plates.

Screening Transformants and Genotyping:

The colonies were picked from dark grown-modified Proteose plates with1% sucrose, and approximately the same amount of cells were transferredto 24 well-plates containing 1 ml of modified Proteose liquid media with1% sucrose. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm for 5 days.

To verify the presence of the invertase gene introduced in Chlorellatransformants, DNA of each transformant was isolated and amplified witha set of gene-specific primers (CMV construct: forward primer(CAACCACGTCTTCAAAGCAA) (SEQ ID NO: 153)/reverse primer(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO: 171), CV constructs: forward primer(TTGTCGGAATGTCATATCAA) (SEQ ID NO: 172)/reverse primer(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO: 171), and HUP1 construct: forwardprimer (AACGCCTTTGTACAACTGCA) (SEQ ID NO: 173)/reverse primer(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO: 171)). For quick DNA isolation, avolume of cells (approximately 5-10 uL in size) were resuspended in 50uL of 10 mM Na-EDTA. The cell suspension was incubated at 100° C. for 10mM and sonicated for 10 sec. After centrifugation at 12000 g for 1 mM, 3uL of supernatant was used for the PCR reaction. PCR amplification wasperformed in the DNA thermal cycler (Perkin-Elmer GeneAmp 9600). Thereaction mixture (50 uL) contained 3 uL extracted DNA, 100 μmol each ofthe respective primers described above, 200 uM dNTP, 0.5 units of TaqDNA polymerase (NEB), and Taq DNA polymerase buffer according to themanufacturer's instructions. Denaturation of DNA was carried out at 95°C. for 5 min for the first cycle, and then for 30 sec. Primer annealingand extension reactions were carried out at 58° C. for 30 sec and 72° C.for 1 min respectively. The PCR products were then visualized on 1%agarose gels stained with ethidium bromide.

Growth in Liquid Culture:

After five days growth in darkness, the genotype-positive transformantsshowed growth on minimal liquid Proteose media+1% sucrose in darkness,while wild-type cells showed no growth in the same media in darkness.

Example 6 Transformation of Algal Strains with a Secreted InvertaseDerived from S. cerevisiae

Secreted Invertase:

A gene encoding a secreted sucrose invertase (Gen Bank Accession no.NP_(—)012104 from Saccharomyces cerevisiae) was synthesized de-novo as a1599 bp Asc I-Xho fragment that was subsequently sub-cloned into a pUC19derivative possessing the Cauliflower Mosaic Virus 35s promoter and 3′UTR as EcoR I/Asc I and Xho/Sac I cassettes, respectively.

Growth of Algal Cells:

Media used in these experiments was liquid base media (2 g/L yeastextract, 2.94 mM NaNO₃, 0.17 mM CaCl₂.2H₂O, 0.3 mM MgSO₄.7H₂O, 0.4 mMK₂HPO₄, 1.28 mM KH₂PO₄, 0.43 mM NaCl) and solid base media (+1.5%agarose) containing fixed carbon in the form of sucrose or glucose (asdesignated) at 1% final concentration. The strains used in thisexperiment did not grow in the dark on base media in the absence of anadditional fixed carbon source. Species were struck out on plates, andgrown in the dark at 28° C. Single colonies were picked and used toinoculate 500 mL of liquid base media containing 1% glucose and allowedto grow in the dark until mid-log phase, measuring cell counts each day.Each of the following strains had been previously tested for growth onsucrose in the dark as a sole carbon source and exhibited no growth, andwere thus chosen for transformation with a secreted invertase: (1)Chlorella protothecoides (UTEX 31); (2) Chlorella minutissima (UTEX2341); and (3) Chlorella emersonii (CCAP 211/15).

Transformation of Algal Cells via Particle Bombardment:

Sufficient culture was centrifuged to give approximately 1−5×10⁸ totalcells. The resulting pellet was washed with base media with no addedfixed carbon source. Cells were centrifuged again and the pellet wasresuspended in a volume of base media sufficient to give 5×10⁷ to 2×10⁸cells/ml. 250-1000 μl of cells were then plated on solid base mediasupplemented with 1% sucrose and allowed to dry onto the plate in asterile hood. Plasmid DNA was precipitated onto gold particles accordingto the manufacturer's recommendations (Seashell Technology, La Jolla,Calif.). Transformations were carried out using a BioRad PDS He-1000particle delivery system using 1350 psi rupture disks with themacrocarrier assembly set at 9 cm from the rupture disk holder.Following transformations, plates were incubated in the dark at 28° C.All strains generated multiple transformant colonies. Control platestransformed with no invertase insert, but otherwise prepared in anidentical fashion, contained no colonies.

Analysis of Chlorella protothecoides Transformants:

Genomic DNA was extracted from Chlorella protothecoides wild type cellsand transformant colonies as follows: Cells were resuspended in 100 ulextraction buffer (87.5 mM Tris Cl, pH 8.0, 50 mM NaCl, 5 mM EDTA, pH8.0, 0.25% SDS) and incubated at 60° C., with occasional mixing viainversion, for 30 minutes. For PCR, samples were diluted 1:100 in 20 mMTris Cl, pH 8.0.

Genotyping was done on genomic DNA extracted from WT, the transformantsand plasmid DNA. The samples were genotyped for the marker gene. Primers2383 (5′ CTGACCCGACCTATGGGAGCGCTCTTGGC 3′) (SEQ ID NO: 174) and 2279 (5′CTTGACTTCCCTCACCTGGAATTTGTCG 3′) (SEQ ID NO: 175) were used in thisgenotyping PCR. The PCR profile used was as follows: 94° C. denaturationfor 5 mM; 35 cycles of 94° C.-30 sec, 60° C.-30 sec, 72° C.-3 mM; 72°C.-5 mM A band of identical size was amplified from the positivecontrols (plasmid) and two transformants of Chlorella protothecoides(UTEX 31).

Analysis of Chlorella minutissima and Chlorella emersonii transformants:Genomic DNA was extracted from Chlorella WT and the tranformants asfollows: Cells were resuspended in 100 ul extraction buffer (87.5 mMTris Cl, pH 8.0, 50 mM NaCl, 5 mM EDTA, pH 8.0, 0.25% SDS) and incubatedat 60° C., with occasional mixing via inversion, for 30 minutes. ForPCR, samples were diluted 1:100 in 20 mM Tris Cl, pH 8.0. Genotyping wasdone on genomic DNA extracted from WT, the transformants and plasmidDNA. The samples were genotyped for the marker gene. Primers 2336 (5′GTGGCCATATGGACTTACAA 3′) (SEQ ID NO: 176) and 2279 (5′CTTGACTTCCCTCACCTGGAATTTGTCG 3′) (SEQ ID NO: 175) were designated primerset 2 (1215 bp expected product), while primers 2465 (5′CAAGGGCTGGATGAATGACCCCAATGGACTGTGGTACGACG 3′) (SEQ ID NO: 177) and 2470(5′ CACCCGTCGTCATGTTCACGGAGCCCAGTGCG 3′) (SEQ ID NO: 178) weredesignated primer set 4 (1442 bp expected product). The PCR profile usedwas as follows: 94° C. denaturation for 2 min; 29 cycles of 94° C.-30sec, 60° C.-30 sec, 72° C.-1 min, 30 sec; 72° C.-5 min. A plasmidcontrol containing the secreted invertase was used as a PCR control.

The sequence of the invertase construct corresponds to SEQ ID NO: 8.

Example 7 Homologous Recombination in Prototheca Species

Homologous recombination of transgenes has several advantages. First,the introduction of transgenes without homologous recombination can beunpredictable because there is no control over the number of copies ofthe plasmid that gets introduced into the cell. Also, the introductionof transgenes without homologous recombination can be unstable becausethe plasmid may remain episomal and is lost over subsequent celldivisions. Another advantage of homologous recombination is the abilityto “knock-out” gene targets, introduce epitope tags, switch promoters ofendogenous genes and otherwise alter gene targets (e.g., theintroduction of point mutations.

Two vectors were constructed using a specific region of the Protothecamoriformis (UTEX 1435) genome, designated KE858. KE858 is a 1.3 kb,genomic fragment that encompasses part of the coding region for aprotein that shares homology with the transfer RNA (tRNA) family ofproteins. Southern blots have shown that the KE858 sequence is presentin a single copy in the Prototheca moriformis (UTEX 1435) genome. Thefirst type of vector that was constructed, designated SZ725 (SEQ ID NO:179), consisted of the entire 1.3 kb KE858 fragment cloned into a pUC19vector backbone that also contains the optimized yeast invertase (suc2)gene. The KE858 fragment contains a unique SnaB 1 site that does notoccur anywhere else in the targeting construct. The second type ofvector that was constructed, designated SZ726 (SEQ ID NO: 180),consisted of the KE858 sequence that had been disrupted by the insertionof the yeast invertase gene (suc2) at the SnaB 1 site within the KE858genomic sequence. The entire DNA fragment containing the KE858 sequencesflanking the yeast invertase gene can be excised from the vectorbackbone by digestion with EcoRI, which cuts at either end of the KE858region.

Both vectors were used to direct homologous recombination of the yeastinvertase gene (suc2) into the corresponding KE858 region of thePrototheca moriformis (UTEX 1435) genome. The linear DNA ends homologousto the genomic region that was being targeted for homologousrecombination were exposed by digesting the vector construct SZ725 withSnaB 1 and vector construct SZ726 with EcoRI. The digested vectorconstructs were then introduced into Prototheca moriformis culturesusing methods described above. Transformants from each vector constructwere then selected using sucrose plates. Ten independent, clonally puretransformants from each vector transformation were analyzed forsuccessful recombination of the yeast invertase gene into the desiredgenomic location (using Southern blots) and for transgene stability.

Southern blot analysis of the SZ725 transformants showed that 4 out ofthe 10 transformants picked for analysis contained the predictedrecombinant bands, indicating that a single crossover event had occurredbetween the KE858 sequences on the vector and the KE858 sequences in thegenome. In contrast, all ten of the SZ726 transformants contained thepredicted recombinat bands, indicating that double crossover events hadoccurred between the EcoRI fragment of pSZ726 carrying KE858 sequenceflanking the yeast invertase transgene and the corresponding KE858region of the genome.

Sucrose invertase expression and transgene stability were assessed bygrowing the transformants for over 15 generations in the absence ofselection. The four SZ725 transformants and the ten SZ276 transformantsthat were positive for the transgene by Southern blotting were selectedand 48 single colonies from each of the transformants were grownserially: first without selection in glucose containing media and thenwith selection in media containing sucrose as the sole carbon source.All ten SZ276 transformants (100%) retained their ability to grow onsucrose after 15 generations, whereas about 97% of the SZ725transformants retained their ability to grow on sucrose after 15generations. Transgenes introduced by a double crossover event (SZ726vector) have extremely high stability over generation doublings. Incontrast, transgenes introduced by a single cross over event (SZ725vector) can result in some instability over generation doublings becauseis tandem copies of the transgenes were introduced, the repeatedhomologous regions flanking the transgenes may recombine and excise thetransgenic DNA located between them.

These experiments demonstrate the successful use of homologousrecombination to generate Prototheca transformants containing aheterologous sucrose invertase gene that is stably integrated into thenuclear chromosomes of the organism. The success of the homologousrecombination enables other genomic alterations in Prototheca, includinggene deletions, point mutations and epitope tagging a desired geneproduct. These experiments also demonstrate the first documented systemfor homologous recombination in the nuclear genome of a eukaryoticmicroalgae.

Use of Homologous Recombination to Knock-Out an Endogenous Protothecamoriformis Gene

In a Prototheca moriformis cDNA/genomic screen, like that describedabove in Example 4, an endogenous stearoyl ACP desaturase (SAPD) cDNAwas identified. Stearoyl ACP desaturase enzymes are part of the lipidsynthesis pathway and they function to introduce double bonds into thefatty acyl chains. In some cases, it may be advantages to knock-out orreduce the expression of lipid pathway enzymes in order to alter a fattyacid profile. A homologous recombination construct was created to assesswhether the expression of an endogenous stearoyl ACP desaturase enzymecan be reduced (or knocked out) and if a corresponding reduction inunsaturated fatty acids can be observed in the lipid profile of the hostcell. An approximately 1.5 kb coding sequence of a stearoyl ACPdesaturase gene from Prototheca moriformis (UTEX 1435) was identifiedand cloned (SEQ ID NO: 181). The homologous recombination construct wasconstructed using 0.5 kb of the SAPD coding sequence at the 5′ end (5′targeting site), followed by the Chlamydomonas reinhardtii β-tublinpromoter driving a codon-optimized yeast sucrose invertase suc2 genewith the Chlorella vulgaris 3′UTR. The rest (−1 kb) of the Protothecamoriformis SAPD coding sequence was then inserted after the C. vulgaris3′UTR to make up the 3′ targeting site. The sequence for this homologousrecombination cassette is listed in SEQ ID NO: 182. As shown above, thesuccess-rate for integration of the homologous recombination cassetteinto the nuclear genome can be increased by linearizing the cassettebefore transforming the microalgae, leaving exposed ends. The homologousrecombination cassette targeting an endogenous SAPD enzyme in Protothecamoriformis is linearized and then transformed into the host cell(Prototheca moriformis, UTEX 1435). A successful integration willeliminate the endogenous SAPD enzyme coding region from the host genomevia a double reciprocal recombination event, while expression of thenewly inserted suc2 gene will be regulated by the C. reinhardtiiβ-tubulin promoter. The resulting clones can be screened usingplates/media containing sucrose as the sole carbon source. Clonescontaining a successful integration of the homologous recombinationcassette will have the ability to grow on sucrose as the sole carbonsource and changes in overall saturation of the fatty acids in the lipidprofile will serve as a secondary confirmation factor. Additionally,Southern blotting assays using a probe specific for the yeast sucroseinvertase suc2 gene and RT-PCR can also confirm the presence andexpression of the invertase gene in positive clones. As an alternative,the same construct without the β-tubulin promoter can be used to excisethe endogenous SAPD enzyme coding region. In this case, the newlyinserted yeast sucrose invertase suc2 gene will be regulated by theendogenous SAPD promoter/5′UTR.

Example 8 Expression of Various Thioesterases in Prototheca

Methods and effects of expressing a heterologous thioesterase gene inPrototheca species have been previously described in PCT Application No.PCT/US2009/66142, hereby incorporated by reference. The effect of otherthioesterase genes/gene products from higher plants species was furtherinvestigated. These thioesterases include thioesterases from thefollowing higher plants:

GenBank Species Accession No. Specificity SEQ ID NO: Cinnamomum Q39473C14 SEQ ID NOs: 30-31 camphora Umbellularia Q41635 C10-C12 SEQ ID NOs:34-35 californica Cuphea hookeriana AAC49269 C8-C10 SEQ ID NOs: 32-33Cuphea palustris AAC49179 C8 SEQ ID NOs: 36-37 Cuphea lanceolataCAB60830 C10 SEQ ID NOs: 38-39 Iris germanica AAG43858.1 C14 SEQ ID NOs:40-41 Myristica fragrans AAB717291.1 C14 SEQ ID NOs: 42-43 Cupheapalustris AAC49180 C14 SEQ ID NOs: 44-45 Ulmus americana AAB71731 broadSEQ ID NOs: 46-47

In all cases, each of the above thioesterase constructs was transformedin to Prototheca moriformis (UTEX 1435) using biolistic particlebombardment. Other transformation methods including homologousrecombination as disclosed in PCT Application No. PCT/US2009/66142,would also be suitable for heterologous expression of genes of interest.Transformation of Prototheca moriformis (UTEX 1435) with each of theabove thioesterase constructs was performed using the methods describedin Example 2. Each of the constructs contained a NeoR gene and selectionfor positive clones was carried out using 100 μg/ml G418. All codingregions were codon optimized to reflect the codon bias inherent inPrototheca moriformis UTEX 1435 (see Table 2) nuclear genes. Both aminoacid sequences and the cDNA sequences for the construct used are listedin the sequence identity listing. The transit peptide for each of thehigher plant thioesterase was replaced with an algal codon optimizedtransit peptide from Prototheca moriformis delta 12 fatty aciddesaturase (SEQ ID NO: 48)) or from Chlorella protothecoides stearoylACP desaturase (SEQ ID NO: 49). All thioesterase constructs were drivenby the Chlamydomanas reinhardtii beta-tubulin promoter/5′UTR. Growth andlipid production of selected positive clones were compared to wildtype(untransformed) Prototheca moriformis (UTEX 1435). Wildtype and selectedpositive clones were grown on 2% glucose G418 plates. Lipid profilesanalysis on selected positive clones for each construct is summarizedbelow (expressed in Area %) in Table 15.

TABLE 15 Lipid profiles of Prototheca moriformis expressing variousheterologous thioesterases. UTEX Thioesterase Fatty 1435 U. C. I. M. C.palustris C. C. C. palustris U. Acid wt californica camphora germanicafragrans C8:0 hookeriana lanceolata C14:0 americana  C8:0 0 0 0 0 3.11.8 0 0 0.9 C10:0 0.02 .07 .02 .01 .09 .56 6.85 1.91 .01 2.85 C12:0 0.0514 1.82 .09 .05 .25 .2 .29 .06 .74 C14:0 1.65 3 17.3 2.59 5.31 1.45 1.81.83 2.87 10.45 C16:0 28.0 21.4 24.3 26.52 31.08 22.84 23.9 25.55 27.2333.3 C18:0 2.9 2.9 2.7 3.11 2.71 3.24 2.8 3.26 3.62 3.47 C18:1 53.8 45.241.3 49.96 39.77 56.62 49.8 55.43 51.04 38.71 C18:2 10.95 10 9.7 11.8614.17 8.24 9.7 8.17 10.81 7.38  C18:3 α 0.8 .86 .8 .40 .64 .61 .9 .58.97 .52 Total 32.62 44.97 46.14 32.32 39.24 31.44 37.35 32.84 33.79 50.9saturates (area %)

The results show that all of the thioesterases expressed impacted fattyacid profiles to some level. Looking at the “Total saturates” row, thedegree of saturation was profoundly impacted by the expression ofseveral of the thioesterases, including those from U. californica, C.camphora, and most notably, U. americana. These changes in thepercentage of total saturates were unexpected in that the heterologousexpression of thioesterases from higher plants can apparently impactmore than just lipid chain lengths; it can also impact other attributesof lipid profiles produced by microalgae, namely the degree ofsaturation of the fatty acids.

Selected clones transformed with C. palustris C8 thioesterase, C.hookeriana thioesterase, U. californica and C. camphora thioesterasewere further grown in varing amounts of G418 (from 25 mg/L to 50 mg/L)and at varying temperatures (from 22° C. to 25° C.) and the lipidprofile was determined for these clones. Table 16 summarizes the lipidprofile (in Area %) of representative clones containing eachthioesterase. A second construct containing the U. americanathioesterase was constructed and transformed into Prototheca moriformis(UTEX 1435) using the biolistic methods described above. This secondconstruct was introduced into the cell via homologous recombination.Methods of homologous recombination in Prototheca species were describedpreviously in PCT Application No. PCT/US2009/66142. The homologous DNAthat was used was from genomic DNA sequence of 6S rRNA from Protothecamoriformis UTEX 1435. The selection agent was the ability to grow onsucrose, using a codon optimized suc2 gene from S. cereveisiae driven bythe C. reinhardtii beta tubulin promoter. The native U. americanatransit peptide was replaced by the Chlorella protothecoides (UTEX 250)stearoyl ACP desaturase transit peptide. The cDNA of this construct islisted in the Sequence Listing as SEQ ID NO: 50. Selection of positiveclones was performed on 2% sucrose plates and the resulting cultures forlipid profile determination was also grown on 2% sucrose containingmedium. A representative lipid profile for this Prototheca moriformisstrain containing a homologously recombined heterologous U. americanathioesterase is summarized in Table 16.

TABLE 16 Lipid profiles of Prototheca moriformis strains containingheterologous thioesterase genes. C. palustris U. americana C8 C.hookeriana C. camphora 2 C8:0 12.28 2.37 0 0 C10:0 2.17 12.09 0.02 4.69C12:0 0.34 0.33 3.81 1.02 C14:0 1.59 2.08 32.73 16.21 C16:0 15.91 20.0724.03 38.39 C18:0 1.59 1.57 1.21 2.83 C18:1 50.64 41.80 18.64 27.22C18:2 13.02 16.37 16.57 7.65 C18:3 α 1.52 1.75 1.66 0.74 Total 33.8838.51 61.80 63.14 saturates

As with the clones described above, all transformants containing aheterologous thioesterase gene showed impacted fatty acid profiles tosome level, and the total percent of saturated fatty acids were alsochanged, as compared to wildtype (untransformed) Prototheca moriformis.The Prototheca moriformis containing the U. americana thioesteraseintroduced by homologous recombination had the greatest increase intotal saturates.

Additionally, transgenic clones containing the exogenous C. hookeriana,C. camphora, U. californica or U. americana thioesterase were assessedfor novel lipid profiles. The C. hookeriana thioesterase containingclone achieved the following lipid profile when grown in 2% glucose, 25mg/ml G418 at 22° C.: 5.10% C8:0; 18.28% C10:0; 0.41% C12:0; 1.76%C14:0; 16.31% C16:0; 1.40% C18:0; 40.49% C18:1; and 13.16% C18:2. The C.camphora thioesterase-containing clone (also containing an exogenoussucrose invertase) achieved the following lipid profile when grown in 2%sucrose at 25° C.: 0.04% C10:0; 6.01% C12:0; 35.98% C14:0; 19.42 C16:0;1.48% C18:0; 25.44% C18:1; and 9.34% C18:2. The U. calfornicathioesterase containing clone achieved the following lipid profile whengrown in 2% glucose, 25-100 mg/ml G418 at 22° C.: 0% C8:0; 0.11% C10:0;34.01% C12:0; 5.75% C14:0; 14.02% C16:0; 1.10% C18:0; 28.93% C18:1; and13.01% C18:2. The U. americana thioesterase containing clone achievedthe following lipid profile when grown in 2% glucose at 28° C.: 1.54%C10:0; 0.43% C12:0; 7.56% C14:0; 39.45% C16:0; 2.49% C18:0; 38.49%C18:1; and 7.88% C18:2.

Example 9 Transformation of Prototheca with Multiple ExogenousHeterologous Thioesterase Genes

Microalgae strain Prototheca moriformis (UTEX 1435) was transformedusing the above disclosed methods to express multiple thioesterases in asingle clone. The expression of multiple thioesterases in a single cloneallows the microaglae to produce oils with fatty acid profilescompletely different from those elaborated when any single thioesteraseis expressed alone (as demonstrated in the preceding Examples).Prototheca moriformis (UTEX 1435) was first transformed with theCinnamomum camphora thioesterase (a C14 preferring thioesterase) alongwith a sucrose invertase gene, the suc2 from S. cerevisiae (selectionwas the ability to grow on sucrose) using homologous recombination. TheDNA used for this homologous recombination construct is from the KE858region of Prototheca moriformis genomic DNA as described in the SectionIII above. The relevant portion of this construct is listed in theSequence Listing as SEQ ID NO: 51. Positive clones were screened onsucrose-containing plates. A positive clone was then re-transformed withone of three cassettes, each encoding resistance to the antibiotic G418as well as an additional thioesterase: (1) thioesterase gene from Cupheahookeriana (C8-10 preferring), SEQ ID NO: 52; (2) thioesterase gene fromUmbellularia californica (C12 preferring), SEQ ID NO: 53; orthioesterase from Ulmus americana (broad; C10-C16 preferring), SEQ IDNO: 54. Included in the Sequence Listing is the sequence of the relevantportion of each construct. Clones expressing both thioesterase geneswere screened on sucrose containing medium with 50 μg/ml G418. Positiveclones were selected and growth and lipid profile were assayed. Table 17summarizes the lipid profile of representative positive clones(expressed in Area %).

TABLE 17 Lipid profiles of Prototheca moriformis transformed withmultiple thioesterases. UTEX 1435 + UTEX C. camphora TE geneticbackground 1435 + +C. +U. +U. Fatty UTEX C. camphora hookerianacalifornica americana Acid 1435 TE TE TE TE C8:0 0 0 0.19 0 0.06 C10:00.02 0.02 2.16 0.07 1.87 C12:0 0.05 0.66 0.53 13.55 1.61 C14:0 1.6510.52 7.64 8.0 14.58 C16:0 28.0 22.56 22.31 19.98 29.53 C18:0 2.9 6.673.23 2.24 2.93 C18:1 53.8 47.78 48.54 42.55 37.3 C18:2 10.95 12.3 11.7610.13 8.9 C18:3 α 0.8 0.93 0.91 0.91 0.76 Total 32.62 40.43 36.06 43.8450.58 saturates (Area %)

Additionally, a double thioesterase clone with C. camphora and U.californica thioesterases was grown in 2% sucrose containing medium with50 mg/L G418 at 22° C. The fatty acid profile obtained from this strainunder these growth conditions was: C8:0 (0); C10:0 (0.10); C12:0(31.03); C14:0 (7.47); C16:0 (15.20); C18:0 (0.90); C18:1 (30.60); C18:2(12.44); and C18:3a (1.38), with a total saturates of 54.7.

Double thioesterase clones with two homologous recombination constructs(one targeting the 6S region and the other targeting the KE858 region)containing the C. camphora thioestease were produced. A positiverepresentative clone had a fatty acid profile of: 0% C8:0; 0.06% C10:0;5.91% C12:0; 43.27% C14:0; 19.63% C16:0; 0.87% C18:0; 13.96% C18:1; and13.78% C18:2, with a total saturates at 69.74%. This clone had a C12-C14level at over 49%, which is over 37 times the C12-C14 level in wildtypecells.

The above data shows that multiple thioesterases can be successfullyco-expressed in microalgae. The co-expression of multiple thioesterasesresults in altered fatty acid profiles that differ significantly notonly from the wild type strain, but also from the fatty acid profileobtained by the expression of any one of the individual thioesterases.The expression of multiple thioesterases with overlapping chain lengthspecificity can result in cumulative increases in those specific fattyacids.

The expression of heterologous thioesterases (either alone or incombination) in Prototheca moriformis not only alters the fattyacid/lipid profiles in the host strain, but when compared to oilscurrently available from a variety of seed crops (Table 5), theseprofiles are of truly unique oils found in no other currently availablesystem. Not only do the transgenic strains show significant differencesfrom the untransformed wildtype strain, they have remarkably differentprofiles from any of the commercial oils that are shown in Table 5. Asan example, both coconut and palm kernel oils have levels of C8-C10fatty acids ranging from 5.5-17%. Transgenic strain expressing the C.palustris C8-preferring thioesterase or the C. hookeriana C10-preferringthioesterase accumulates anywhere from 3.66 to 8.65%, respectively.These C8-C10 fatty acid levels are similar to coconut oil and palmkernel, however, the transgenic algal strains lack the significantlyhigher C12:0 fatty acids, and they have extremely high C16:0 (23% intransgenics versus 11-16% in coconut or palm kernel oil, respectivelyand/or 18:1 (50-57% in transgenics versus 8-19% in coconut or palmkernel oil, respectively.

Example 10 Identification of Endogenous Nitrogen-Dependent ProtothecaPromoters

A. Identification and Characterization of Endogenous Nitrogen-DependentPromoters.

A cDNA library was generated from Prototheca moriformis (UTEX 1435)using standard techniques. The Prototheca moriformis cells were grownfor 48 hours under nitrogen replete conditions. Then a 5% innoculum(v/v) was then transferred to low nitrogen and the cells were harvestedevery 24 hours for seven days. After about 24 hours in culture, thenitrogen supply in the media was completely depleted. The collectedsamples were immediately frozen using dry ice and isopropanol. Total RNAwas subsequently isolated from the frozen cell pellet samples and aportion from each sample was held in reserve for RT-PCR studies. Therest of the total RNA harvested from the samples was subjected to polyAselection. Equimolar amounts of polyA selected RNA from each conditionwas then pooled and used to generate a cDNA library in vector pcDNA 3.0(Invitrogen). Roughly 1200 clones were randomly picked from theresulting pooled cDNA libray and subjected to sequencing on bothstrands. Approximately 68 different cDNAs were selected from among these1200 sequences and used to design cDNA-specific primers for use inreal-time RT-PCR studies.

RNA isolated from the cell pellet samples that were held in reserve wasused as substrate in the real time RT-PCR studies using thecDNA-specific primer sets generated above. This reserved RNA wasconverted into cDNA and used as substrate for RT-PCR for each of the 68gene specific primer sets. Threshold cylcle or C_(T) numbers were usedto indicate relative transcript abundance for each of the 68 cDNAswithin each RNA sample collected throughout the time course. cDNAsshowing significant increase (greater than three fold) between nitrogenreplete and nitrogen-depleted conditions were flagged as potential geneswhose expression was up-regulated by nitrogen depletion. As discussed inthe specification, nitrogen depletion/limitation is a known inducer oflipogenesis in oleaginous microorganisms.

In order to identify putative promoters/5′UTR sequences from the cDNAswhose expression was upregulated during nitrogen depletion/limitation,total DNA was isolated from Prototheca moriformis (UTEX 1435) grownunder nitrogen replete conditions and were then subjected to sequencingusing 454 sequencing technology (Roche). cDNAs flagged as beingup-regulated by the RT-PCR results above were compared using BLASTagainst assembled contigs arising from the 454 genomic sequencing reads.The 5′ ends of cDNAs were mapped to specific contigs, and wherepossible, greater than 500 bp of 5′ flanking DNA was used to putativelyidentify promoters/UTRs. The presence of promoters/5′UTR weresubsequently confirmed and cloned using PCR amplification of genomicDNA. Individual cDNA 5′ ends were used to design 3′ primers and 5′ endof the 454 contig assemblies were used to design 5′ gene-specificprimers.

As a first screen, one of the putative promoters, the 5′UTR/promoterisolated from Aat2 (Ammonium transporter, SEQ ID NO: 63), was clonedinto the Cinnamomum camphora C14 thioesterase construct with theChlorella protothecoides stearoyl ACP desaturase transit peptide,replacing the C. sorokinana glutamate dehydrogenase promoter. Thisconstruct is listed as SEQ ID NO: 81. To test the putative promoter, thethioesterase construct is transformed into Prototheca moriformis cellsto confirm actual promoter activity by screening for an increase inC14/C12 fatty acids under low/no nitrogen conditions, using the methodsdescribed above. Similar testing of the putative nitrogen-regulatedpromoters isolated from the cDNA/genomic screen can be done using thesame methods.

Other putative nitrogen-regulated promoters/5′UTRs that were isolatedfrom the cDNA/genomic screen were:

Fold Promoter/5′UTR SEQ ID NO. increased FatB/A promoter/5′UTR SEQ IDNO: 55 n/a NRAMP metal transporter promoter/ SEQ ID NO: 56 9.65 5′UTRFlap Flagellar-associated protein promoter/ SEQ ID NO: 57 4.92 5′UTRSulfRed Sulfite reductase promoter/5′UTR SEQ ID NO: 58 10.91 SugT Sugartransporter promoter/5′UTR SEQ ID NO: 59 17.35 Amt03-Ammoniumtransporter 03 SEQ ID NO: 60 10.1 promoter/5′UTR Amt02-Ammoniumtransporter 02 SEQ ID NO: 61 10.76 promoter/5′UTR Aat01-Amino acidtransporter 01 SEQ ID NO: 62 6.21 promoter/5′UTR Aat02-Amino acidtransporter 02 SEQ ID NO: 63 6.5 promoter/5′UTR Aat03-Amino acidtransporter 03 SEQ ID NO: 64 7.87 promoter/5′UTR Aat04-Amino acidtransporter 04 SEQ ID NO: 65 10.95 promoter/5′UTR Aat05-Amino acidtransporter 05 SEQ ID NO: 66 6.71 promoter/5′UTR

Fold increase refers to the fold increase in cDNA abundance after 24hours of culture in low nitrogen medium.

To gain further insight into potential regulation of these putativepromoter/5′UTRs, eight of the sequences were selected for furthertesting: (1) FatB/A; (2) SulfRed Sulfite reductase; (3) SugT Sugartransporter; (4) Amt02-Ammonium transporter 02; (5) Aat01-Amino acidtransporter 01; (6) Aat03-Amino acid transporter 03; (7) Aat04-Aminoacid transporter 04; and (8) Aat05-Amino acid transporter 05. Higherresolution transcriptome analysis utilizing Illumina sequencing readswere carried out on RNA isolated from Prototheca moriformis cellsvarious time points: TO (seed); 20 hours; 32 hours; 48 hours; 62 hours;and 114 hours post inoculation from seed. The medium at T0 (seed) wasnitrogen replete, while at the time points 20 hours and longer, themedium contained little to no nitrogen. Assembled transcript contigsgenerated from RNA isolated from each of the time points were thenblasted independently with each of the eight previously identifiedtranscripts. The results are summarized in Table 18 below.

TABLE 18 Transcriptome expression profiles for eight putativepromoters/5'UTRs. cDNA TS T20 T32 T48 T62 T114 aa trans_01 absolute 9896 321 745 927 1300 relative 1 0.98 3.28 7.61 9.47 13.28 aa trans_03absolute 7 21 51 137 102 109 relative 1 2.95 7.2 19.42 14.47 15.45 aatrans_04 absolute 1 6 25 90 131 160 relative 1 5.16 21.29 74.97 109.35133.31 aa trans_05 absolute 109 88 123 210 214 273 relative 1 0.81 1.131.93 1.97 2.51 ammon trans_02 absolute 683 173 402 991 1413 1397relative 1 0.25 0.59 1.45 2.07 2.04 fatA/B-1_cDNA absolute 13 36 654 617544 749 relative 1 2.8 51.57 48.65 42.9 59.1 sug trans_01 absolute 25 25106 261 266 251 relative 1 1 4.22 10.4 10.63 10 sulfite reductase 0_1absolute 634 238 138 145 163 155 relative 1 0.38 0.22 0.22 0.26 0.24

From the above-summarized results, several of the transcripts showincreased accumulation over time, although interestingly, the sulfitereductase mRNA shows a distinct decrease in mRNA accumulation over time.

These eight putative promoter/5′UTR regions were cloned upstream of theC. camphora thioesterase coding region with its native transit peptidetaken out and substituted with the transit peptide from Chlorellaprotothecoides (UTEX 250) stearoyl ACP desaturase. Each putativepromoter/5′UTR region construct was introduced into Protothecamoriformis UTEX 1435 via homologous recombination using DNA from thegenomic sequence of 6S rRNA. Also contained within the construct is asuc2 sucrose invertase gene from S. cerevisiae for selection of positiveclones on sucrose containing media/plates. The cDNA sequence for therelevant portions of the construct for Aat01 is listed in the SequenceListing as SEQ ID NO: 67. For the other constructs, the same backbonewas use, the only variable was the putative promoter/5′UTR sequence. Anadditional control transgenic strain was generated in which the C.reinhardtii beta tubulin promoter was used to drive expression of the C.camphora thioesterase gene. This promoter have shown to driveconstitutive expression of the gene of interest, and thus provides auseful control against which to measure expression of the samethioesterase message when driven by the various putative N-regulatedpromoters/5′UTRs tested.

Once the transgenic clones were generated, three separate experimentswere carried out. The first two experiments assess the potentialnitrogen regulatability of all eight putative promoters by measuringsteady state thioesterase mRNA levels via RT-PCR, fatty acid profilesand ammonia levels in the culture supernatants. Clones were initiallygrown at 28° C. with agitation (200 rpm) in nitrogen rich seed medium (1g/L ammonium nitrate-15 mM nitrogen as ammonia, 4 g/L yeast extract) for24 to 48 hours, at which point 20 OD units (A₇₅₀) were used to inoculate50 ml of low nitrogen media (0.2 g/L ammonium sulfate-3 mM nitrogen asammonia, 0.2 g/L yeast extract). Cells were sampled every 24 hours for 6days and a sample was also collected right before switching to lownitrogen conditions. A portion of the cells from each sample was thenused for total RNA extraction using Trizol reagent (according tomanufacturer's suggested methods) Ammonia assays revealed that ammonialevels in the supernatants fell below the limits of detection (˜100 μM)after 24 hours in low nitrogen medium.

For real-time RT-PCR, all RNA levels were normalized to levels of aninternal control RNA expressed in Prototheca moriformis (UTEX 1435) foreach time point. The internal control RNA, termed cd189, is a product ofthe ARG9 gene which encodes N-acetyl ornithine aminotransferase. Primerssets used for real-time RT-PCR in these experiments were:

Gene specific to Primer sequence 5′-3′ SEQ ID NO: C. camphoraTACCCCGCCTGGGGCGACAC SEQ ID NO: 68 TE forward C. camphoraCTTGCTCAGGCGGCGGGTGC SEQ ID NO: 69 TE reverse cd189 forwardCCGGATCTCGGCCAGGGCTA SEQ ID NO: 70 cd189 reverse TCGATGTCGTGCACCGTCGCSEQ ID NO: 71

Lipid profiles from each of the transformants from each time point werealso generated and compared to the RT-PCR results. Based on the ammonialevels, RT-PCR results and changes in C12-C14 fatty acid levels, it wasconcluded that the Amino acid transporter 01 (Aat-01), Amino acidtransporter 04 (Aat-04), and Ammonium transporter 02 (Amt-02) sequencesdo contain a functional nitrogen-regulatable promoter/5′UTR.

From the RT-PCR results, Aat-01 demonstrated the ability to drive steadystate C. camphora thioesterase mRNA levels up to four times higher thancontrol (C. reinhardtii beta tubulin promoter). The mRNA levels alsocorrelated with nitrogen limitation and a marked increase in C12-C14fatty acid levels. These results demonstrate that the 5′UTR associatedwith the Aat-01 promoter is likely more efficient at driving proteinsynthesis under lipid biosynthesis than the control C. reinhardtiipromoter. Like the Aat-01 promoter, the Aat-04 promoter was able todrive mRNA accumulation up to five times higher than that of the C.reinhardtii control promoter. However, the Aat-04 promoter constructonly produced a modest ability to impact C12-C14 fatty acid levels.These data demonstrate that the Aat-04 promoter is clearly regulatableby nitrogen depletion, but the UTR associated with the promoter likelyfunctions poorly as a translational enhancer. Finally, the Amt-02promoter was similar to the Aat-01 promoter, in that it was able todrive mRNA accumulation up to three times higher than that of thecontrol promoter. The mRNA levels also correlated with nitrogenlimitation and a marked increase in C12-C14 fatty acid levels. Takentogether, all three of these promoters were demonstrated to benitrogen-regulated.

B. Further Characterization of the Ammonium Transporter 3 (amt03)Promoter and Expression of Various Thioesterases.

As described above, partial cDNAs termed ammonium transporter 02 and 03(amt02 and amt03) were identified. Along with these two partial cDNAs, athird partial cDNA termed ammonium transporter 01 (amt01) was alsoidentified. Alignment of the partial cDNA and the putative translatedamino acid sequences were compared. Results show amt01 to be moredistantly related of the three sequences, while amt02 and amt03 differby only a single amino acid.

Promoters/5′UTRs were generated initially in silico by blasting thepartial cDNA sequences against Roche 454 genomic DNA assemblies andIllumina transcriptome assemblies as described above. Transcript contigsshowing identity to the cDNA encoding amt01, amt02, and amt03 wereidentified, however, the transcript contigs could not differentiatebetween the three mRNAs as the contigs contained sequences shared by allthree. Roche 454 genomic DNA assemblies gave hits to amt02 and amt03cDNA sequences and contained N-terminal protein sequences. PCR wascarried out to clone the 5′ flanking regions. The PCR primers used tovalidate the clone amt02 and amt03 promoter/UTR were:

Amt03 forward: (SEQ ID NO: 85) 5′-GGAGGAATTCGGCCGACAGGACGCGCGTCA-3′Amt03 reverse: (SEQ ID NO: 86) 5′-GGAGACTAGTGGCTGCGACCGGCCTGTG-3′Amt02 forward: (SEQ ID NO: 87) 5′-GGAGGAATTCTCACCAGCGGACAAAGCACCG-3′Amt02 reverse: (SEQ ID NO: 88) 5′-GGAGACTAGTGGCTGCGACCGGCCTCTGG-3′In both cases, the 5′ and 3′ primers contained useful restriction sitesfor the anticipated cloning into expression vectors to validate thefunctionality of these promoter/5′UTR regions.

Pair wise alignments between the DNAs cloned through this combined insilico and PCR-based method and the original cDNA encoding amt02 (SEQ IDNO: 61) and amt03 (SEQ ID NO: 60) were performed. Results of thesealignments showed significant differences between the original cDNAs andthe cloned genomic sequences, indicating that ammonium transporterslikely represent a diverse gene family. Additionally, the promoter/5′UTRclone based on the combined method for amt03 was different than theoriginal amt03 sequence, whereas the amt02 sequences were identical.Further experiments to characterize the amt03 promoter/UTR sequence (SEQID NO: 89) was carried out and described below.

The above identified amt03 promoter/UTR sequence (SEQ ID NO: 89) wastested by cloning this putative promoter/UTR sequence to drive theexpression of four different thioesterases. The expression cassettecontained upstream and downstream homologous recombination sequences tothe 6S locus of the genome (SEQ ID NOs: 82 and 84, respectively). Thecassette also contains a S. cerevisiae SUC2 sucrose invertase cDNA toenable the selection for positive clones on sucrose containing medium.The sucrose invertase expression was driven by the C. reinhardtii betatubulin promoter and also contained a C. vulgaris nitrate reductase3′UTR. The amt03 promoter/UTR sequence was then cloned downstream of thesucrose invertase cassette followed by in-frame thioesterase cDNAsequence from one of four thioesterase genes: (1) C14 thioesterase fromC. camphora; (2) C12 thioesterase from U. californica; (3) C10-C16thioesterase from U. americana; or (4) C10 thioesterase from C.hookeriana and also contained a C. vulgaris nitrate reductase 3′UTR. TheC14 C. camphora thioesterase, C12 U. californica thioesterase, and theC10-C16 U. americana all contained the transit peptide from a Chlorellaprotothecoides stearoyl ACP desaturase. The C10 C. hookerianathioesterase contained the transit peptide from a Prototheca moriformisdelta 12 fatty acid desaturase (FAD). In all cases, the sequences werecodon optimized for expression in Prototheca moriformis. The sequencesto the foregoing thioesterase constructs are described in the SequenceListing:

amt03 promoter/UTR::C. camphora SEQ ID NO: 90 thioesterase construct C.camphora thioesterase construct SEQ ID NO: 91 U. californicathioesterase construct SEQ ID NO: 92 U. americana thioesterase constructSEQ ID NO: 93 C. hookeriana thioesterase construct SEQ ID NO: 94

Transgenic lines were generated via biolistic transformation methods asdescribed above in Example 2 into wild type Prototheca moriformis cellsand selection was carried out on sucrose containing plates/medium.Positive lines were then screened for the degree to which their fattyacid profiles were altered. Four lines, one resulting from thetransformation with each of the four above-described constructs, werethen subjected to additional analysis. Line 76 expressed the C. camphoraC14 thioesterase, line 37 expressed the U. californica C12 thioesterase,line 60 expressed the U. americana C10-C16 thioesterase, and line 56expressed the C. hookeriana C10 thioesterase. Each line was grown for 48hours in medium containing sucrose as the sole carbon source and samplesof cells were removed at 14, 24, 36 and 48 hours (seed culture) fordetermination of fatty acid profile via direct transesterification tofatty acid methyl esters and subsequent analysis by GC-FID (describedabove) and for isolation of total RNA. At the end of 48 hours, thesecells were used to inoculate cultures with no or low levels of nitrogen(containing sucrose as the sole carbon source) maintained at either pH5.0 (citrate buffered, 0.05M final concentration) or pH 7.0 (HEPESbuffered, 0.1M final concentration). Culture samples were removed at 12,24, 72 and 108 hours (lipid production) for fatty acid profiling andisolation of total RNA Ammonia assays of these cultures revealed thatammonia levels fell below the limits of detection (ca. 100 μM) after 24hours in low nitrogen medium.

Real-time RT-PCR assays on the mRNA levels of the thioesterases wereperformed on total RNA from each of the time points collected above andall mRNA levels were normalized to the levels of an internal control RNA(cd189). Primer sets used in real-time PCR are shown in Table 19 below:

TABLE 19 Primer sets for real-time PCR. Gene specific toPrimer sequence 5′-3′ SEQ ID NO: C. camphora TACCCCGCCTGGGGCGACACSEQ ID NO: TE forward 68 C. camphora CTTGCTCAGGCGGCGGGTGC SEQ ID NO:TE reverse 69 U. californica CTGGGCGACGGCTTCGGCAC SEQ ID NO: TE forward95 U. californica AAGTCGCGGCGCATGCCGTT SEQ ID NO: TE reverse 96U. americana CCCAGCTGCTCACCTGCACC SEQ ID NO: TE forward 97 U. americanaCACCCAAGGCCAACGGCAGCGCCGTG SEQ ID NO: TE reverse 98 C. hookerianaTACCCCGCCTGGGGCGACAC SEQ ID NO: TE forward 99 C. hookerianaAGCTTGGACAGGCGGCGGGT SEQ ID NO: TE reverse 100 cd189 reverseTCGATGTCGTGCACCGTCGC SEQ ID NO: 71 cd189 forward CCGGATCTCGGCCAGGGCTASEQ ID NO: 70

The results from the fatty acid profiles at each of the time points inthe seed culture phase showed very little impact from the thioesterases.With the commencement of the lipid production phase, the fatty acidprofiles were significantly impacted, with the increases that are farmore dramatic for the cultures maintained at pH 7.0 as compared to thecultures at pH 5.0. While the magnitude of the difference between pH 7.0and 5.0 target fatty acid accumulation varied with each thioesterasetested, the overall effect was the same: that the cells grown at pH 5.0showed significantly lower levels of the target fatty acids accumulated,but more than compared to control wild type cells.

Analysis of the RNA isolated from these same samples correlated verywill with the fatty acid profile data, in that there was a clear impactof culture pH on the steady state mRNA levels for each of thethioesterases. Taking the fatty acid accumulation data and the mRNA datatogether, the pH regulation of thioesterase gene expression driven bythe amt03 promoter/UTR was clearly mediated either at the level oftranscription, mRNA stability or both. Additionally, it was observedthat the steady state levels of U. californica mRNA were four logs loweras compared to the steady state levels of C. hookeriana mRNA. Thisobservation is consistent with the hypothesis that the individual mRNAsequences may play a role in controlling expression. These data implythat ammonium uptake in Prototheca moriformis by the amt03 family oftransporters is coupled directly to pH.

Additional fatty acid profile analysis was performed on twelve linesgenerated from the transformation of Prototheca moriformis cells withthe construct amt03 promoter/UTR driving the expression of the U.americana C10-C16 thioesterase. Line 60, described above, was a part ofthe following analysis. Table 20 below shows the lipid profiles of threeof the twelve lines that were analyzed along with the wild type control.

TABLE 20 Fatty acid profiles of transformants containing the U.americana TE driven by the amt03 promoter/UTR. Total Sat- Area% C8:0C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 urates wild type 0.00 0.010.04 1.27 27.20 3.85 58.70 7.18 32.36 Line 40 2.38 20.61 3.41 28.4129.92 1.91 8.57 3.74 86.64 Line 44 1.50 20.16 4.44 31.88 26.66 1.88 6.955.42 86.50 Line 60 0.98 14.56 3.15 27.49 31.76 2.14 12.23 6.36 80.06

As shown in the table above, the levels of total saturates was increaseddramatically over that of wild type with over 2.6 fold in the case ofline 40 compared to wildtype (total saturates from the twelve linesanalyzed ranged from about 63% to over 86%). Additionally, the U.americana thioesterase, when expressed at these levels, dramaticallyreduces the level of unsaturates, especially C18:1 and C18:2 (see lines40 and 44), where in line 44, C18:1 levels are reduced by over 8 foldcompared to the wild type. Also, the U. americana thioesterase (drivenby the amt03 promoter) greatly increases the levels of mid-chain fattyacids. Line 44 shows C10:0-C14:0 levels at greater than 56%,approximately 42 fold higher than the levels seen in the wildtype strainand C8:0-C14:0 levels at greater than 57%. Additional strainstransformed with a construct of the Amt03 promoter driving theexpression of the U. americana thioesterase had representative lipidprofile of: 0.23% C8:0; 9.64% C10:0; 2.62% C12:0; 31.52% C14:0; 37.63%C16:0; 5.34% C18:0; 7.05% C18:1; and 5.03% C18:2, with a total saturatespercentage at 86.98%.

Additional lipid profiles generated from the transformation ofPrototheca moriformis cells with the construct amt03 promoter/UTR (SEQID NO: 89) driving the expression of the C. hookeriana C10 thioesterase(SEQ ID NO: 94). Positive clones expressing this construct were selectedand grown at pH 7.0 conditions. Representative lipid profile from apositive clone was: 9.87% C8:0; 23.97% C10:0; 0.46% C12:0; 1.24% C14:0;10.24% C16:0; 2.45% C18:0; 42.81% C18:1; and 7.32% C18:2. This clone hada C8-C10 percentage of 33.84

Taken together, the data suggest that the amt03 promoter/UTR, and otherpromoters like it, can be used as a tightly regulated promoter, whichmay be particularly useful for expressing a potentially toxic compoundand strict enforcement of gene expression is required. The ability ofPrototheca moriformis to grow under a wide range (at least pH 5.0 to7.0) of pH regimes makes this organism particularly useful incombination with regulatory elements such as the amt03 promoter/UTR.Additionally, the lipid profile data above demonstrates the impressiveability of the amt03 promoter/UTR to drive gene expression.

Example 11 Altering the Levels of Saturated Fatty Acids in theMicroalgae Prototheca moriformis

As part of a genomics screen using a bioinformatics based approach basedon cDNAs, Illumia transcriptome and Roche 454 squencing of genomic DNAfrom Prototheca moriformis (UTEX 1435), two specific groups of genesinvolved in fatty acid desaturation were identified: stearoyl ACPdesaturases (SAD) and delta 12 fatty acid desaturases (Δ12 FAD).Stearoyl ACP desaturase enzymes are part of the lipid synthesis pathwayand they function to introduce double bonds into the fatty acyl chains,for example, the synthesis of C18:1 fatty acids from C18:0 fatty acids.Delta 12 fatty acid desaturases are also part of the lipid synthesispathway and they function to introduce double bonds into alreadyunsaturated fatty acids, for example, the synthesis of C18:2 fatty acidsfrom C18:1 fatty acids. Southern blot analysis using probes based on thetwo classes of fatty acid desaturase genes identified during thebioinformatics efforts indicated that each class of desaturase genes waslikely comprised of multiple family members. Additionally the genesencoding stearoyl ACP desaturases fell into two distinct families Basedon these results, three gene disruption constructs were designed topotentially disrupt multiple gene family members by targeting morehighly conserved coding regions within each family of desaturaseenzymes.

Three homologous recombination targeting constructs were designed using:(1) highly conserved portions of the coding sequence of delta 12 fattyacid desaturase (d12FAD) family members and (2) two constructs targetingeach of the two distinct families of SAD, each with conserved regions ofthe coding sequences from each family. This strategy would embed aselectable marker gene (the suc2 sucrose invertase cassette from S.cerevisiae conferring the ability to hydrolyze sucrose) into thesehighly conserved coding regions (targeting multiple family members)rather than a classic gene replacement strategy where the homologousrecombination would target flanking regions of the targeted gene.

All constructs were introduced into the cells by biolistictransformation using the methods described above and constructs werelinearized before being shot into the cells. Transformants were selectedon sucrose containing plates/media and changes in lipid profile wereassayed using the above-described method. Relevant sequences from eachof the three targeting constructs are listed below.

Description SEQ ID NO: 5′ sequence from coding region of d12FAD from SEQID NO: 72 targeting construct 3′ sequence from coding region of d12FADfrom SEQ ID NO: 73 targeting construct d12FAD targeting construct cDNAsequence SEQ ID NO: 74 5′ sequence from coding region of SAD2A SEQ IDNO: 75 3′ sequence from coding region of SAD2A SEQ ID NO: 76 SAD2Atargeting construct cDNA sequence SEQ ID NO: 77 5′ sequence from codingregion os SAD2B SEQ ID NO: 78 3′ sequence from coding region of SAD2BSEQ ID NO: 79 SAD2B targeting construct cDNA sequence SEQ ID NO: 80

Representative positive clones from transformations with each of theconstructs were picked and the lipid profiles for these clones weredetermined (expressed in Area %) and summarized in Table 21 below.

TABLE 21 Lipid profiles for desaturase knockouts. Fatty Acid d12FAD KOSAD2A KO SAD2B KO wt UTEX 1435 C8:0 0 0 0 0 C10:0 0.01 0.01 0.01 0.01C12:0 0.03 0.03 0.03 0.03 C14:0 1.08 0.985 0.795 1.46 C16:0 24.42 25.33523.66 29.87 C18:0 6.85 12.89 19.555 3.345 C18:1 58.35 47.865 43.11554.09 C18:2 7.33 10.27 9.83 9.1 C18:3 alpha 0.83 0.86 1 0.89 C20:0 0.480.86 1.175 0.325

Each of the construct had a measurable impact on the desired class offatty acid and in all three cases C18:0 levels increased markedly,particularly with the two SAD knockouts. Further comparison of multipleclones from the SAD knockouts indicated that the SAD2B knockout lineshad significantly greater reductions in C18:1 fatty acids than the C18:1fatty acid levels observed with the SAD2A knockout lines.

Additional Δ12 fatty acid desaturase (FAD) knockouts were generated in aPrototheca moriformis background using the methods described above. Inorder to identify potential homologous of Δ12FADs, the following primerswere used in order to amplify a genomic region encoding a putative FAD:

SEQ ID NO: 101 Primer 1 5′-TCACTTCATGCCGGCGGTCC-3′ SEQ ID NO: 102Primer 2 5′-GCGCTCCTGCTTGGCTCGAA-3′

The sequences resulting from the genomic amplification of Protothecamoriformis genomic DNA using the above primers were highly similar, butindicated that multiple genes or alleles of Δ12FADs exist in Prototheca.

Based on this result, two gene disruption constructs were designed thatsought to inactivate one or more Δ12FAD genes. The strategy would toembed a sucrose invertase (suc2 from S. cerevisiae) cassette, thusconferring the ability to hydrolyze sucrose as a selectable marker, intohighly conserved coding regions rather than use a classic genereplacement strategy. The first construct, termed pSZ1124, contained 5′and 3′ genomic targeting sequences flanking a C. reinhardtii β-tubulinpromoter driving the expression of the S. cerevisiae suc2 gene and aChlorella vulgaris nitrate reductase 3′UTR (S. cerevisiae suc2cassette). The second construct, termed pSZ1125, contained 5′ and 3′genomic targeting sequences flanking a C. reinhardtii β-tubulin promoterdriving the expression of the S. cerevisiae suc2 gene and a Chlorellavulgaris nitrate reductase 3′UTR. The relevant sequences of theconstructs are listed in the Sequence Listing:

pSZ1124 (FAD2B) 5′ genomic targeting sequence SEQ ID NO: 103 pSZ1124(FAD2B) 3′ genomic targeting sequence SEQ ID NO: 104 S. cerevisiae suc2cassette SEQ ID NO: 105 pSZ1125 (FAD2C) 5′ genomic targeting sequenceSEQ ID NO: 106 pSZ1125 (FAD2C) 3′ genomic targeting sequence SEQ ID NO:107

pSZ1124 and pSZ1125 were each introduced into a Prototheca moriformisbackground and positive clones were selected based on the ability tohydrolyze sucrose. Table 22 summarizes the lipid profiles (in Area %,generated using methods described above) obtained in two transgeniclines in which pSZ1124 and pSZ1125 targeting vectors were utilized.

TABLE 22 Lipid profiles of Δ12 FAD knockouts C10:0 C12:0 C14:0 C16:0C16:1 C18:0 C18:1 C18:2 C18:3α parent 0.01 0.03 1.15 26.13 1.32 4.3957.20 8.13 0.61 FAD2B 0.02 0.03 0.80 12.84 1.92 0.86 74.74 7.08 0.33FAD2C 0.02 0.04 1.42 25.85 1.65 2.44 66.11 1.39 0.22

The transgenic containing the FAD2B (pSZ1124) construct gave a veryinteresting and unexpected result in lipid profile, in that the C18:2levels, which would be expected to decrease, only decreased by about onearea %. However, the C18:1 fatty acid levels increased significantly,almost exclusively at the expense of the C16:0 levels, which decreasedsignificantly. The transgenic containing the FAD2C (pSZ1125) constructalso gave a change in lipid profile: the levels of C18:2 are reducedsignificantly along with a corresponding increase in C18:1 levels.

Beef Tallow Mimetic

One positive clone generated from the above SAD2B knockout experiment asdescribed above was selected to be used as the background for thefurther introduction of a C14-preferring fatty acyl-ACP thioesterasegene. The construct introducing the C. camphora C14-preferringthioesterase contained targeting sequence to the 6S rRNA genomic region(allowing for targeted integration of the transforming DNA viahomologous recombination) and the expression construct contained the C.reinhardtii β-tubulin promoter driving the expression of the neoR genewith the Chlorella vulgaris nitrate reductase 3′UTR, followed by asecond C. reinhardtii β-tubulin promter driving the expression of acodon-optimized C. camphora thioesterase with a Chlorella protothecoidesstearoyl ACP desaturase transit peptide with a second Chlorella vulgarisnitrate reductase 3′UTR. The 5′ 6S rRNA genomic donor sequence is listedin SEQ ID NO: 82; the 3′ 6S rRNA genomic donor sequence is listed in SEQID NO: 84; and the relevant expression construct for the C. camphorathioesterase is listed in SEQ ID NO: 83.

Transformation was carried out using biolistic methods as describedabove and the cells were allowed to recover for 24 hours on platescontaining 2% sucrose. After this time, the cells were re-suspended andre-plated on plates containing 2% sucrose and 50 μg/ml G418 forselection. Nine clones out of the positive clones generated wereselected for lipid production and lipid profile. The nine transgenicclones (with the SAD2B KO and expressing C. camphora C14-preferringthioesterase) were cultured as described above and analyzed for lipidprofile. The results are summarized below in Table 23. The lipid profilefor tallow is also included in Table 23 below (National Research Council1976: Fat Content and Composition of Animal Product).

TABLE 23 Lipid profile of thioesterase transformed clones. C10:0 C12:0C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20 SAD2BKO 0.01 0.33 6.1324.24 0.19 11.08 42.03 13.45 0.98 0.73 C.camphora TE clone 1 SAD2BKO0.01 0.16 3.42 23.80 0.40 9.40 50.62 10.2 0.62 0.70 C.camphora TE clone2 SAD2BKO 0.01 0.20 4.21 25.69 0.40 7.79 50.51 9.37 0.66 0.63 C.camphoraTE clone 3 SAD2BKO 0.01 0.21 4.29 23.57 0.31 9.44 50.07 10.07 0.70 0.70C.camphora TE clone 4 SAD2BKO 0.01 0.18 3.87 24.42 0.32 9.24 49.75 10.170.71 0.71 C.camphora TE clone 5 SAD2BKO 0.01 0.28 5.34 23.78 0.33 9.1249.12 10.00 0.68 0.70 C.camphora TE clone 6 SAD2BKO 0.01 0.15 3.09 23.070.32 10.08 51.21 10.00 0.66 0.74 C.camphora TE clone 7 SAD2BKO 0.01 0.295.33 24.62 0.37 7.02 49.67 10.74 0.69 0.70 C.camphora TE clone 8 SAD2BKO0.01 0.12 2.74 25.13 0.30 10.17 50.18 9.42 0.71 0.71 C.camphora TE clone9 wt UTEX 0.01 0.02 0.96 23.06 0.79 3.14 61.82 9.06 0.46 0.27 1435SAD2BKO 0.01 0.03 0.80 23.66 0.13 19.56 43.12 9.83 1.00 1.18 Tallow 0.000.00 4.00 26.00 3.00 14.00 41.00 3.00 1.00 0.00

As can be seen in Table 23, the lipid profiles of the transgenic linesare quite similar to the lipid profile of tallow. Taken collectively,the data demonstrate the utility of combining specific transgenicbackgrounds, in this case, a SAD2B knockout with a C14-preferringthioesterase (from C. camphora), to generate an transgenic algal strainthat produce oil similar to the lipid profile of tallow.

Construct Used to Down Regulate the Expression of β-Ketoacyl Synthase II(KASII) by Targeted Knock-Out Approach

Vector down-regulating KASII gene expression by targeted knock-outapproach was introduced into a classically mutagenized derivative ofUTEX 1435, S1331. The Saccharomyces cerevisiae invertase gene wasutilized as a selectable marker, conferring the ability to grow onsucrose. The invertase expression cassette under control of C.reinhardtii B-tubulin promoter was inserted in the middle of the 315 bplong KASII genomic region to permit targeted integration (pSZ1503).

Relevant restriction sites in pSZ1503 are indicated in lowercase, boldand underlining and are 5′-3′ BspQ 1, Kpn I, AscI, Xho I, Sac I, BspQ I,respectively. BspQI sites delimit the 5′ and 3′ ends of the transformingDNA. Bold, lowercase sequences represent genomic DNA from S1331 thatpermit targeted integration at KASII locus via homologous recombination.Proceeding in the 5′ to 3′ direction, the C. reinhardtii B-tubulinpromoter driving the expression of the yeast sucrose invertase gene(conferring the ability of S1331 to metabolize sucrose) is indicated byboxed text. The initiator ATG and terminator TGA for invertase areindicated by uppercase, bold italics while the coding region isindicated in lowercase italics. The Chlorella vulgaris nitrate reductase3′ UTR is indicated by lowercase underlined text.

Nucleotide sequence of transforming DNA contained inpSZ1503_[KASII_btub-y.inv-nr_KASII]:

(SEQ ID NO: 149) gctcttcccgcaccggctggctccaccccaacttgaacctcgagaaccccgcgcctggcgtcgacccgtcgtgctcgtggggccgcggaaggagcgcgccgaagacctggacgtcgtcctctccaactcctttggctttggcgggcacaattcgtgcgtcggtacc

ccggcttcgccgccaagatcagcgcctccatgacgaacgagcgtccgaccgccccctggtgcacttcaccccaacaaggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgaccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaactcgacctgtccaacagcaccggcacctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcgtagagctcatcttccgaaagtacgacgagtgagcgagctgattctctttgagcggggtcgggtggttcggggagagtgcgcggaaaggcgcagagacgtgcggccggccgtgtccctccgtcttcccctggttggtgctatagtaacctgcctgtgtcgcgtgcgcgtcgg gaagagc

The cDNAs of the KAS II allele 1 and allele 2 are identified in SEQ IDNOs: 279 and 280, respectively. The amino acid sequences of alleles 1and 2 are identified in SEQ ID NOs: 281 and 282, respectively.

To determine the impact of KASII inactivation on lipid composition,pSZ1503 vector DNA was transformed into S1331 to generate a targetedKASII knock-out phenotype. Initial single clones were isolated and grownunder standard lipid production conditions at pH5.0. The resultingprofiles of the best representative clone and the wild-type cells areshown below in Table 23A.

TABLE 23A Fatty acid profiles in S1331 and a derivative transgenic linetransformed with pSZ1503 DNA. Sample ID C10:0 C12:0 C14:0 C16:0 C16:1C18:0 C18:1 C18:2 C18:3 α 1331-5 0.01 0.03 0.96 24.28 0.64 3.94 62.696.21 0.49 D698-2 0.01 0.01 0.83 38.36 1.38 2.21 48.31 7.60 0.55

Example 12 Engineering Prototheca with Alternative Selectable Markers

A. Expression of a Secretable α-Galactosidase in Prototheca moriformis

Methods and effects of expressing a heterologous sucrose invertase genein Prototheca species have been previously described in PCT ApplicationNo. PCT/US2009/66142, hereby incorporated by reference. The expressionof other heterologous polysaccharide degrading enzymes was examined inthis Example. The ability to grow on melibiose (α-D-gal-glu) byPrototheca moriformis UTEX 1435 with one of the following exogenous geneencoding a α-galactosidase was tested: MEL1 gene from Saccharomycescarlbergensis (amino acid sequence corresponding to NCBI accessionnumber P04824 (SEQ ID NO: 108)), AglC gene from Aspergillus niger (aminoacid sequence corresponding to NCBI accession number Q9UUZ4 (SEQ ID NO:116)), and the α-galactosidase from the higher plant Cyamopsistetragobobola (Guar bean) (amino acid sequence corresponding to NCBIaccession number P14749 (SEQ ID NO: 120). The above accession numbersand corresponding amino acid sequences are hereby incorporated byreference. In all cases, genes were optimized according to the preferredcodon usage in Prototheca moriformis. The relevant portions of theexpression cassette are listed below along with the Sequence Listingnumbers. All expression cassettes used the 5′ and 3′ Clp homologousrecombination targeting sequences for stable genomic integration, theChlamydomonas reinhardtii TUB2 promoter/5′UTR, and the Chlorellavulgaris nitrate reductase 3′UTR.

S. carlbergensis MEL1 amino acid sequence SEQ ID NO: 108 S.carlbergensis MEL1 amino acid sequence signal SEQ ID NO: 109 peptide S.carlbergensis MEL1 transformation cassette SEQ ID NO: 110 S.carlbergensis MEL1 sequence (codon optimized) SEQ ID NO: 111 5′ Clphomologous recombination targeting sequence SEQ ID NO: 112 3′ Clphomologous recombination targeting sequence SEQ ID NO: 113 Chlamydomonasreinhardtii TUB2 promoter/5′UTR SEQ ID NO: 114 Chlorella vulgarisnitrate reductase 3′UTR SEQ ID NO: 115 A. niger AlgC amino acid sequenceSEQ ID NO: 116 A. niger AlgC amino acid sequence signal peptide SEQ IDNO: 117 A. niger AlgC sequence (codon optimized) SEQ ID NO: 118 A. nigerAlgC transformation cassette SEQ ID NO: 119 C. tetragonobolaα-galactosidase amino acid sequence SEQ ID NO: 120 C. tetragonobolaα-galactosidase sequence SEQ ID NO: 121 (codon optimized) C.tetragonobola α-galactosidase transformation SEQ ID NO: 122 cassette

Prototheca moriformis cells were transformed with each of the threeexpression cassettes containing S. carlbergensis MEL1, A. niger AlgC, orC. tetragonobola α-galactosidase gene using the biolistic transformationmethods as described in Example 2 above. Positive clones were screenedusing plates containing 2% melibiose as the sole carbon source. Nocolonies appeared on the plates for the C. tetragonobola expressioncassette transformants. Positive clones were picked from the platescontaining the S. carlbergensis MEL1 transformants and the A. niger AlgCtransformants. Integration of the transforming DNA was confirmed usingPCR with primers targeting a portion of the C. vulgaris 3′UTR and the 3′Clp homologous recombination targeting sequence.

5′ primer C. vulgaris 3′UTR: downstream Clp sequence (SEQ ID NO: 123)ACTGCAATGCTGATGCACGGGA 3′ primer C. vulgaris 3′UTR: downstream Clpsequence (SEQ ID NO: 124) TCCAGGTCCTTTTCGCACT

As a negative control, genomic DNA from untransformed Protothecamoriformis cells were also amplified with the primer set. No productswere amplified from genomic DNA from the wild type cells.

Several positive clones from each of the S. carlbergensis MEL1transformants and the A. niger AlgC transformants (as confirmed by PCR)were tested for their ability to grow on melibiose as the sole carbonsource in liquid media. These selected clones were grown for 3 days inconditions and base medium described in Example 1 above with melibioseas the sole carbon source. All clones containing eitherα-galactosidase-encoding genes grew robustly during this time, while theuntransformed wild type strain and Prototheca moriformis expressing aSaccharomyces cerevisiae SUC2 sucrose invertase both grew poorly on themelibiose media. These results suggest that the α-galactosidase encodinggenes may be used as a selectable marker for transformation. Also, thesedata indicate that the native signal peptides present in the S.carlbergensis MEL1 (SEQ ID NO: 109) or A. niger AlgC (SEQ ID NO: 117)are useful for targeting proteins to the periplasm in Protothecamoriformis cells.

B. THIC Genes Complements Thiamine Auxotrophs in Prototheca

Thiamine prototrophy in Prototheca moriformis cells was examined usingexpression of exogenous THIC genes. Thiamine biosynthesis in plants andalgae is typically carried out in the plastid, hence most nuclearencoded proteins involved in its production will need to be efficientlytargeted to the plastid. DNA sequencing and transcriptome sequencing ofPrototheca moriformis cells revealed that all of the genes encoding thethiamine biosynthetic enzymes were present in the genome, with theexception of THIC. To dissect the lesion responsible for thiamineauxotrophy at the biochemical level, the growth of Prototheca moriformiscells under five different regimes were examined: (1) in the presence of2 μM thiamine hydrochloride; (2) without thiamine; (3) without thiamine,but with 2 μM hydroxyethyl thiazole (THZ); (4) without thiamine, butwith 2 μM 2-methyl-4-amino-5-(aminomethyl)pyrimidine (PYR); and (5)without thiamine, but with 2 μM THZ and 2 μM PYR. Results from thegrowth experiments under these 5 different conditions indicated thatPrototheca moriformis cells are capable of de novo synthesis, but canonly produce thiamine pyrophosphate (TPP) if the PYR precursor isprovided. This result is consistent with the hypothesis that thethiamine auxotrophy of Prototheca moriformis is due to the inability tosynthesize hydroxymethylpyrimidine phosphate (HMP-P) from aminoimidazoleribonucleotide, which is the conversion catalyze by THIC enzyme.

Prototheca moriformis cells were transformed using the biolistictransformation methods described above in Example 2, expressing theCoccomyxa C-169 THIC (amino acid sequence corresponding to JGI ProteinID 30481, and hereby incorporated by reference) and a S. cerevisiae SUC2sucrose invertase as the selective marker. This expression constructcontained the native transit peptide sequence from Coccomyxa C-169 THIC,upstream and downstream homologous recombination targeting sequences tothe 6S region of genomic DNA, a C. reinhardtii TUB2 promoter/5′UTRregion (SEQ ID NO: 104), and a Chlorella vulgaris nitrate reductase3′UTR (SEQ ID NO: 115). The S. cerevisiae SUC2 expression was alsodriven by a C. reinhardtii TUB2 promoter/5′UTR region (SEQ ID NO: 114)and contained a Chlorella vulgaris nitrate reductase 3′UTR (SEQ ID NO:115). Genes were optimized according to the preferred codon usage inPrototheca moriformis. The relevant expression cassette sequences arelisted in the Sequence Listing and detailed below:

Coccomyxa C-169 THIC amino acid sequence SEQ ID NO: 125 Coccomyxa C-169THIC amino acid sequence native SEQ ID NO: 126 transit peptide CoccomyxaC-169 THIC transformation cassette SEQ ID NO: 127 Coccomyxa C-169 THICsequence (codon optimized) SEQ ID NO: 128 S. cerevisiae SUC2 sequence(codon optimized) SEQ ID NO: 129 5′ 6S homologous recombinationtargeting sequence SEQ ID NO: 82 3′ 6S homologous recombinationtargeting sequence SEQ ID NO: 84Selection of positive clones were performed on plates without thiamineand containing sucrose as the sole carbon source. Positive clones wereconfirmed using PCR with a 5′ primer that binds within the CoccomyxaC-169 THIC gene and a 3′ primer that anneals downsteam of thetransforming DNA in the 6S locus. PCR confirmed positive clones werealso confirmed using Southern blot assays.

To observe the thiamine auxotrophy of wildtype Prototheca moriformiscells, it was necessary to first deplete cells of internal thiaminereserves. To test growth in medium without thiamine, cells were firstgrown to stationary phase in medium containing 2 μM thiamine and thenthe cells were diluted to an optical density at 750 nm (OD750) ofapproximately 0.05 in medium without thiamine. The diluted cells werethen grown once more to stationary phase in medium without thiamine(about 2-3 days). These thiamine-depleted cells were used to inoculatecultures for growth studies in medium without thiamine. Wildtype cellswere grown in medium with glucose as the carbon source (with or withoutthiamine) and positive clones with the native transit peptide CoccomyxaC-169 THIC construct were grown in medium with sucrose as the solecarbon source. Growth was measured by monitoring the absorbance at 750nm. Results of the growth experiments showed substantial greater growthin thiamine-free medium of strains expressing the transgene compared towildtype cells in thiamine-free medium. However, the transformantsfailed to achieve the growth rate and cell densities of wildtype cellsin thiamine-containing media. There was also a strong correlationbetween the amount of growth in the transformant clones in thiamine-freemedium and the copy number of the integrated Coccomyxa enzyme (i.e., themore copy numbers of the transgene, the better the growth of the cellsin thiamine-free medium).

Additional transformants were generated using expression constructscontaining the Coccomyxa THIC, the Arabidopsis thaliana THIC gene, andthe Synechocystis sp. PCC 6803 thiC gene. In the case of the Coccomyxaand the A. thaliana THIC gene, the native transit peptide sequence wasreplaced with the transit peptide sequence from a Chlorellaprotothecoides stearoyl-ACP desaturase (SAD) gene. Synechocystis sp. isa cyanobacterium and the thiC protein does not contain a native transitpeptide sequence. In the Synechocystis sp thiC construct, the transitpeptide sequence from a Chlorella protothecoides SAD gene was fused tothe N-terminus of the Synechocystis sp. thiC. In all cases, thesequences were codon optimized for expression in Prototheca moriformis.All three of the foregoing constructs contained a upstream anddownstream homologous recombination targeting sequence to the 6S regionof the genome (SEQ ID NOs: 82 and 84), a Chlorella protothecoides actinpromoter/5′ UTR, and a Chlorella protothecoides EFTA gene 3′UTR. Allthree constructs contained a neoR gene driven by the C. reinhardtii TUB2promoter/5′UTR (SEQ ID NO: 114) and contained the C. vulgaris 3′UTR (SEQID NO: 115), conferring the selection by G418. The amino acid sequenceof the A. thaliana THIC corresponded to NCBI accession numberNP_(—)180524 and the amino acid sequence of the Synechocystis sp. thiCcorresponded to NCBI accession number NP_(—)442586, both sequenceshereby incorporated by reference. The relevant expression cassettesequences are listed in the Sequence Listing and detailed below:

Coccomyxa THIC expression construct with SEQ ID NO: 130 C.protothecoides transit peptide Coccomyxa THIC with C. protothecoidestransit SEQ ID NO: 131 peptide C. protothecoides actin promoter/5′ UTRSEQ ID NO: 132 C. protothecoides EF1A 3′UTR SEQ ID NO: 133 A. thalianaTHIC expression construct SEQ ID NO: 134 A. thaliana THIC with C.protothecoides transit SEQ ID NO: 135 peptide A. thaliana THIC aminoacid sequence with native SEQ ID NO: 136 transit peptide Synechocystissp. thiC expression construct SEQ ID NO: 137 Synechocystis sp. thiC withC. protothecoides transit SEQ ID NO: 138 peptide Synechocystis sp. thiCamino acid sequence SEQ ID NO: 139 neoR gene SEQ ID NO: 140

Positive clones were screened on plates containing G418 and severalclones from each transformation were picked for verification by PCR.Integration of the transforming DNA constructs containing the CoccomyxaC-169 (with C. protothecoides transit peptide), A. thaliana andSynechocystis sp. PCC 6803 THIC genes, respectively into the 6S locus ofthe genome was confirmed using PCR analysis with the following primers:

5′ THIC Coccomyxa confirmation primer sequence (SEQ ID NO: 141)ACGTCGCGACCCATGCTTCC 3′ THIC confirmation primer sequence(SEQ ID NO: 142) GGGTGATCGCCTACAAGA 5′THIC A. thaliana confirmation primer sequence (SEQ ID NO: 143)GCGTCATCGCCTACAAGA 5′ thiC Synechocystis sp. confirmation primersequence (SEQ ID NO: 144) CGATGCTGTGCTACGTGA

Growth experiments on thiamine depleted cells (as described above) wereperformed using selected confirmed positive clones from transformants ofeach of the different constructs in medium containing G418. Alltransformants were able to grow (with varying degrees of robustness) inthiamine-free medium. Comparison of the growth of the transformants inthiamine-free medium to wild type cells on thiamine-containing mediumshowed the following ranking with respect to their ability to supportgrowth in thiamine-free medium: (1) A. thaliana transformants; (2)Coccomyxa C-169 (with C. protothecoides transit peptide) transformants;and (3) Synechocystis sp. transformants. These results suggest thatwhile a single copy of A. thaliana THIC was able to complement thiamineauxotrophy in Prototheca moriformis cells, multiple copies of CoccomyxaC-169 (with either the native transit peptide sequence or a transitpeptide sequence from C. protothecoides) and Synechocystis sp. THIC wasrequired to enable rapid growth in the absence of thiamine. Given thevariability in results of the different THIC from the different sources,the ability of any particular THIC gene to fully complement the lesionpresent in Prototheca species is not predictable.

An alignment of the three THIC amino acid sequences was performed. Whilethere exist significant sequence conservation between thiC fromSynechocystis sp. compared to the THICs from Coccomyxa and A. thaliana(41% identity at the amino acid level), the cyanobacterial protein ismissing a domain at the N-terminus that is well-conserved in the algaland plant proteins. Despite the missing domain (and presumably resultingin structural differences), the construct expressing the Synechocystissp. thiC was able to at least partially restore thiamine prototrophic inPrototheca moriformis cells.

Example 13 Fuel Production

A. Extraction of Oil from Microalgae Using an Expeller Press and a PressAid

Microalgal biomass containing 38% oil by DCW was dried using a drumdryer resulting in resulting moisture content of 5-5.5%. The biomass wasfed into a French L250 press. 30.4 kg (67 lbs.) of biomass was fedthrough the press and no oil was recovered. The same dried microbialbiomass combined with varying percentage of switchgrass as a press aidwas fed through the press. The combination of dried microbial biomassand 20% w/w switchgrass yielded the best overall percentage oilrecovery. The pressed cakes were then subjected to hexane extraction andthe final yield for the 20% switchgrass condition was 61.6% of the totalavailable oil (calculated by weight). Biomass with above 50% oil drycell weight did not require the use of a pressing aid such asswitchgrass in order to liberate oil. Other methods of extraction of oilfrom microalgae using an expeller press are described in PCT ApplicationNo. PCT/US2010/31108 and is hereby incorporated by reference.

B. Production of Biodiesel from Prototheca Oil

Degummed oil from Prototheca moriformis UTEX 1435, produced according tothe methods described above, was subjected to transesterification toproduce fatty acid methyl esters. Results are shown in Table 24 below.

The lipid profile of the oil was:

-   C10:0 0.02-   C12:0 0.06-   C14:0 1.81-   C14.1 0.07-   C16:0 24.53-   C16:1 1.22-   C18:0 2.34-   C18:1 59.21-   C18:2 8.91-   C18:3 0.28-   C20:0 0.23-   C20:1 0.10-   C20:1 0.08-   C21:0 0.02-   C22:0 0.06-   C24:0 0.10

TABLE 24 Biodiesel profile from Prototheca moriformis triglyceride oil.Method Test Result Units ASTM Cold Soak Filterability of Filtration Time120 sec D6751 A1 Biodiesel Blend Fuels Volume Filtered 300 ml ASTM D93Pensky-Martens Closed Cup Procedure Used A Flash Point Corrected Flash165.0 ° C. Point ASTM Water and Sediment in Middle Sediment and Water0.000 Vol % D2709 Distillate Fuels (Centrifuge Method) EN 14538Determination of Ca and Mg Sum of (Ca and <1 mg/kg Content by ICP OESMg) EN 14538 Determination of Ca and Mg Sum of (Na and K) <1 mg/kgContent by ICP OES ASTM D445 Kinematic/Dynamic Kinematic Viscosity 4.873mm²/s Viscosity @ 104° F./40° C. ASTM D874 Sulfated Ash from LubricatingSulfated Ash <0.005 Wt % Oils and Additives ASTM Determination of TotalSulfur Sulfur, mg/kg 1.7 mg/kg D5453 in Light Hydrocarbons, SparkIgnition Engine Fuel, Diesel Engine Fuel, and Engine Oil by UltravioletFluorescence. ASTM D130 Corrosion - Copper Strip Biodiesel-Cu 1aCorrosion 50° C. (122° F.)/3 hr ASTM Cloud Point Cloud Point 6 ° C.D2500 ASTM Micro Carbon Residue Average Micro <0.10 Wt % D4530 MethodCarbon Residue ASTM D664 Acid Number of Petroleum Procedure Used AProducts by Potentiometric Acid Number 0.20 mg Titration KOH/g ASTMDetermination of Free and Free Glycerin <0.005 Wt % D6584 Total Glycerinin B-100 Total Glycerin 0.123 Wt % Biodiesel Methyl Esters By GasChromatography ASTM Additive Elements in Phosphorus 0.000200 Wt % D4951Lubricating Oils by ICP-AES ASTM Distillation of Petroleum IBP 248 ° C.D1160 Products at Reduced Pressure AET @ 5% 336 ° C. Recovery AET @ 10%338 ° C. Recovery AET @ 20% 339 ° C. Recovery AET @ 30% 340 ° C.Recovery AET @ 40% 342 ° C. Recovery AET @ 50% 344 ° C. Recovery AET @60% 345 ° C. Recovery AET @ 70% 347 ° C. Recovery AET @ 80% 349 ° C.Recovery AET @ 90% 351 ° C. Recovery AET @ 95% 353 ° C. Recovery FBP 362° C. % Recovered 98.5 % % Loss 1.5 % % Residue 0.0 % Cold Trap Volume0.0 ml IBP 248 ° C. EN 14112 Determination of Oxidation OxidationStability >12 hr Stability (Accelerated Operating Temp 110 ° C.Oxidation Test) (usually 110 deg C.) ASTM Density of Liquids by DigitalAPI Gravity @ 60° F. 29.5 °API D4052 Density Meter ASTM D6890Determination of Ignition Derived Cetane >61.0 Delay (ID) and DerivedNumber (DCN) Cetane Number (DCN)

The lipid profile of the biodiesel was highly similar to the lipidprofile of the feedstock oil. Other oils provided by the methods andcompositions of the invention can be subjected to transesterification toyield biodiesel with lipid profiles including (a) at least 4% C8-C14;(b) at least 0.3% C8; (c) at least 2% C10; (d) at least 2% C12; and (3)at least 30% C8-C14.

The Cold Soak Filterability by the ASTM D6751 A1 method of the biodieselproduced was 120 seconds for a volume of 300 ml. This test involvesfiltration of 300 ml of B100, chilled to 40° F. for 16 hours, allowed towarm to room temp, and filtered under vacuum using 0.7 micron glassfiber filter with stainless steel support. Oils of the invention can betransesterified to generate biodiesel with a cold soak time of less than120 seconds, less than 100 seconds, and less than 90 seconds.

C. Production of Renewable Diesel

Degummed oil from Prototheca moriformis UTEX 1435, produced according tothe methods described above and having the same lipid profile as the oilused to make biodiesel in this Example, above, was subjected totransesterification to produce renewable diesel.

The oil was first hydrotreated to remove oxygen and the glycerolbackbone, yielding n-paraffins. The n-parrafins were then subjected tocracking and isomerization. A chromatogram of the material is shown inFIG. 1. The material was then subjected to cold filtration, whichremoved about 5% of the C18 material. Following the cold filtration thetotal volume material was cut to flash point and evaluated for flashpoint, ASTM D-86 distillation distribution, cloud point and viscosity.Flash point was 63° C.; viscosity was 2.86 cSt (centistokes); cloudpoint was 4° C. ASTM D86 distillation values are shown in Table 25:

Table 25. ASTM D86 distillation values.

TABLE 25 ASTM D86 distillation values. Readings in ° C.: VolumeTemperature IBP 173  5 217.4 10 242.1 15 255.8 20 265.6 30 277.3 40283.5 50 286.6 60 289.4 70 290.9 80 294.3 90 300 95 307.7 FBP 331.5

The T10-T90 of the material produced was 57.9° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other T10-T90 ranges, such as 20, 25,30, 35, 40, 45, 50, 60 and 65° C. using triglyceride oils producedaccording to the methods disclosed herein.

The T10 of the material produced was 242.1° C. Methods of hydrotreating,isomerization, and other covalent modification of oils disclosed herein,as well as methods of distillation and fractionation (such as coldfiltration) disclosed herein, can be employed to generate renewablediesel compositions with other T10 values, such as T10 between 180 and295, between 190 and 270, between 210 and 250, between 225 and 245, andat least 290.

The T90 of the material produced was 300° C. Methods of hydrotreating,isomerization, and other covalent modification of oils disclosed herein,as well as methods of distillation and fractionation (such as coldfiltration) disclosed herein can be employed to generate renewablediesel compositions with other T90 values, such as T90 between 280 and380, between 290 and 360, between 300 and 350, between 310 and 340, andat least 290.

The FBP of the material produced was 300° C. Methods of hydrotreating,isomerization, and other covalent modification of oils disclosed herein,as well as methods of distillation and fractionation (such as coldfiltration) disclosed herein, can be employed to generate renewablediesel compositions with other FBP values, such as FBP between 290 and400, between 300 and 385, between 310 and 370, between 315 and 360, andat least 300.

Other oils provided by the methods and compositions of the invention canbe subjected to combinations of hydrotreating, isomerization, and othercovalent modification including oils with lipid profiles including (a)at least 4% C8-C14; (b) at least 0.3% C8; (c) at least 2% C10; (d) atleast 2% C12; and (3) at least 30% C8-C14.

Example 14 Production of Tailored Oils

Using the methods and materials as disclosed herein, various tailoredoils were produced. Table 32 shows the strain, the gene and the genbankaccession numbers of the genes conferring the phenotype and the variousfatty acid profiles produced by the indicated strain. Strains A and Bare both Prototheca moriformis (UTEX 1435) strains, both of which wereclassically mutagenized by a fee-for-service laboratory to improve oilyield. Strains A and B were then genetically engineered as describedherein with the appropriate DNA constructs to express the desired genes.The strains were also engineered to inactivate endogenous desaturases,as indicated. The nucleotide sequences of the thioesterases were codonoptimized for expression and use in Prototheca.

The fatty acid profile of wild type, un-engineered Prototheca is shownin the first line of Table 32. As can be seen, the fatty acid profilehas been dramatically altered in different ways in the differentstrains. For example, the percentage of C8:0 produced by non-geneticallyengineered P. moriformis cells is 0%. However, P. moriformis cellsengineered to express a C. hookeriana thioesterase increased C8:0production from 0% to 13.2% of the total triglycerides. As anotherexample, the total combined amount of C8:0 and C10:0 in the engineeredstrains was about 39% of the total fatty acids. In contrast, the totalcombined amount of C8:0 and C10:0 in the wild type cells is 0.01%. Inanother example, the total amount of saturated fatty acids was increasedfrom about 32% to about 90% by the expression of an U. americanathioesterase in cells in which expression of endogenous SAD2b wasdisrupted. This is an increase of almost 300%.

The various fatty acid profiles as disclosed below are useful in myriadapplications involving triglyceride oils. For example, high levels oflower carbon chain length saturated fatty acids comprising triglyceride(C12:0, C14:0, C16:0) are particularly useful in renewable jet fuelproduction. For biodiesel production, high amounts of C18:1 aredesirable. For bar soap production, controlling and achieving theappropriate balance between the levels of saturation and shorter chainfatty acids is desirable. As an example, high amounts of C12:0 aredesirable for lathering properties while longer chain lengths providemore structure, while linoleic and linolenic containing triglyceridesare less desirable as they contribute to oxidative instability. Forliquid soaps, high amounts of C12:0 and C14:0 are desirable.Additionally, for both bar soap and liquid soap production, low amountsof C6:0, C8:0 and C10:0 are desirable as these lower chain triglyceridesare skin irritants.

TABLE 25A Genes and accession numbers conferring phenotypes of varioustriglyceride profiles. Gen Bank Accession Gene Conferring Strain GeneticTotal Trait and Description Phenotype Construct* Seq. Id. No. BackgroundC8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Saturates Wild Type naUTEX 1435 0.00 0.01 0.04 1.27 27.20 3.85 58.70 7.18 32.36 Highest C8U39834 C. hookeriana TE pSZ 1458 A 13.20 25.84 0.51 1.41 10.22 1.3938.21 7.42 52.57 Highest C10 U39834 C. hookeriana TE pSZ 1458 A 13.2025.84 0.51 1.41 10.22 1.39 38.21 7.42 52.57 Highest C12 U56104 and C.wrightii TE pSZ 1491 B .02 13.63 50.59 6.49 6.64 0.87 13.74 6.83 78.00U67317 + C. wrightii (SEQ ID NO: 232) (SEQ ID NO: 185) KASA1 Highest C14U31813 Cinnamomum pSZ 941 UTEX 1435 0.00 0.06 5.91 43.27 19.63 0.8713.96 13.78 69.74 camphora TE (SEQ ID NO: 236)/944 (SEQ ID NO: 228)Highest C16 Q39513.1 C. hookeriana TE pSZ 1417 A 0.00 0.02 0.11 10.6269.92 2.18 12.95 5.15 80.35 (SEQ ID NO: 226) Highest C18 U56104 as C.wrightii TE pSZ 1410 A 0.00 0.11 1.28 1.82 24.55 37.38 23.51 7.88 65.14SAD2B gene disruption (SEQ ID NO: 230) Highest C8-C10 U39834 C.hookeriana TE pSZ 1458 A 13.20 25.84 0.51 1.41 10.22 1.39 38.21 7.4252.57 Highest C8-C14 U56104 C. wrightii TE pSZ 1283 A .22 17.64 45.8510.94 5.55 0.79 13.49 4.68 74.65 (SEQ ID NO: 229) Highest C10-C14 U56104C. wrightii TE pSZ 1283 A .22 17.64 45.85 10.94 5.55 0.79 13.49 4.6874.65 (SEQ ID NO: 229) Highest C12-C14 ABB71579.1 C. callophylla TE pSZ1570 B .01 0.88 28.04 34.08 19.82 1.00 10.52 4.42 83.83 (SEQ ID NO: 286)(SEQ ID NO: 235) Lowest 18:1 AAB71731 Ulmus pSZ 1321 A .12 10.39 3.5535.21 33.54 4.90 5.15 5.69 87.71 (SEQ ID NO: 287) americana TE (SEQ IDNO: 242) as SAD2B gene disruption Highest 18:1 FADc Disruption CarthamuspSZ 1500 A 0 0 0 0 16.49 0 83.51 0.00 16.49 with Carthamustincorus TEtinctorus TE (SEQ ID NO: 233) AAA33019.1 Lowest 18:2 FADc DisruptionCarthamus pSZ 1501 A 0 0 .03 1.05 18.01 1.44 77.11 0.00 20.53 withCarthamustincorus TE tinctorus TE (SEQ ID NO: 234) AAA33019.1 HighestAAB71731 Ulmus pSZ 1321 A .30 13.07 3.57 33.58 33.52 5.16 5.36 4.5089.20 Saturates as a SAD2B Disruption americana TE (SEQ ID NO: 242)Palm Kernel Oil

We produced a microbial palm kernel oil mimetic that was similar to palmkernel oil (PKO). To produce the palm kernel oil mimetic, a plasmid wasconstructed and used to transform Strain A and oil production wascarried out. The construct, pSZ1413 (SEQ ID NO: 231), comprised codonoptimized Cuphea wrightii FATB2 gene (SEQ ID NO: 284) (Gen bankaccession no. U56106) and SAD2B (stearoyl ACP desaturase) genedisruption.

As shown in Table 25B below, the palm kernel oil mimetic was similar topalm kernel oil. The percentages of the three most abundant fatty acidsof the PKO mimetic (C12:0, C14:0 and C18:1) were identical to or within10% of the palm kernel oil.

TABLE 25B Triglyceride profile of palm kernel oil mimetic. E. guineensis(Palm C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 kernel) 3.0-5.02.5-6.0 40-52 14.0-18.0 7.0-10.0 1.0-3.0 11.0-19.0 0.5-4.0 pSZ1413 8.3337.45 18.22 13.52 1.25 15.29 4.95Palm Oil

We produced a microbial palm oil mimetic that was similar to palm oil.Several different plasmids were constructed and transformed individuallyinto Strain A and oil production was carried out. The construct, pSZ1503(SEQ ID NO: 283), was designed to disrupt an endogenous KASII gene. Theconstruct, pSZ1439 (SEQ ID NO: 237), comprised a codon optimized Elaeisguiniensis TE gene (SEQ ID NO: 205) (Gen bank accession no. AAD42220.2).The construct, pSZ1420 (SEQ ID NO: 225), comprised a codon optimizedCuphea hookeriana TE gene (SEQ ID NO: 201) (Gen Bank Accession no.Q39513). The construct, pSZ1119 (SEQ ID NO: 227), comprised a codonoptimized Cuphea hookeriana KAS IV gene (SEQ ID NO: 186) (Gen BankAccession no. AF060519) as well as a Cuphea wrightii FATB2 gene (SEQ IDNO: 184) (Gen Bank Accession no. U56104).

As shown in Table 25C below, the palm oil mimetic was similar to palmoil. The percentages of the three most abundant fatty acids of the palmoil mimetic (C16:0, C18:1 and C18:2) were identical to or within 10% ofpalm oil.

TABLE 25C Triglyceride profile of palm oil mimetic. E. guineensis C10:0C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 (Palm) 0 0 0.5-5.9 32.0-47.0 2.0-8.034-44 7.2-12.0 pSZ1503 0.01 0.01 0.83 38.36 2.21 48.31 7.60 pSZ1439 0.010.04 1.88 43.50 3.32 39.95 9.16 pSZ1420 0.02 0.04 2.44 48.04 2.76 35.628.91 pSZ1119 1.77 0.40 7.85 35.45 2.47 42.85 8.15Cocoa Butter

We produced a microbial cocoa butter mimetic that was similar to cocoabutter. The construct, pSZ1451, was constructed and transformed intoStrain A and oil production was carried out. The construct, pSZ1451 (SEQID NO: 239), comprised codon optimized Carthamus tinctorus TE gene (SEQID NO: 187) (Gen Bank Accession no. AAA33019.1).

As shown in Table 25D below, the cocoa butter oil mimetic was similar tococoa butter. The percentages of the three most abundant fatty acids ofthe cocoa butter mimetic (C16:0, C18:0 and C18:1) were identical to orwithin 10% of cocoa butter.

TABLE 25D Triglyceride profile of cocoa butter mimetic. C8:0 C10:0 C12:0C14:0 C16:0 C18:0 C18:1 C18:2 Cocoa Butter 0 0-1 0-1 0-4 22-38 24-3729-38 0-3 pSZ1451 0.05 0.14 0.99 28.34 27.39 29.40 10.26Lard

We produced a microbial lard mimetic that was similar to lard. Severaldifferent plasmids were constructed and transformed individually intoStrain A and oil production was carried out. The construct, pSZ1493 (SEQID NO: 241), was designed to disrupt the endogenous SAD 2B gene andsimultaneously express a codon optimized Umbellularia californica TEgene (SEQ ID NO: 285) (Gen Bank Accession no. M94159). The construct,pSZ1452 (SEQ ID NO: 240), was designed to disrupt the endogenous SAD 2Bgene and express a codon optimized Garcinia mangostana TE gene (SEQ IDNO: 196) (Gen Bank Accession no. AAB51525.1). The construct, pSZ1449(SEQ ID NO: 238), was designed to express the codon optimized Brassicanapus TE gene (SEQ ID NO: 195) (Gen Bank Accession no. CAA52070.1). Thepolynucleotide sequence of the construct pSZ1458 was identical topSZ1449 except that a codon optimized polynucleotide sequence encoding aCuphea hookeriana thioesterase (Gen Bank accession No. U39834) replacedthe polynucleotide sequence encoding Brassica napus TE gene (SEQ ID NO:195) (Gen Bank Accession no. CAA52070.1).

As shown in Table 36 below, the lard mimetic was similar to lard. Thepercentages of the three most abundant fatty acids of the lard mimetic(C16:0, C18:0 and C18:1) were identical to or within 10% of lard.

TABLE 36 Triglyceride profile of lard mimetic. C14:0 C16:0 C18:0 C18:1C18:2 Lard 3-4 22-26 13-18 39-45 8-15 pSZ1493 1.32 24.79 17.49 41.8710.01 pSZ1452 1.16 24.49 17.94 45.49 8.05 pSZ1449 1.16 23.98 15.79 47.888.29

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

All references cited herein, including patents, patent applications, andpublications, including Genbank Accession numbers, are herebyincorporated by reference in their entireties, whether previouslyspecifically incorporated or not. The publications mentioned herein arecited for the purpose of describing and disclosing reagents,methodologies and concepts that may be used in connection with thepresent invention. Nothing herein is to be construed as an admissionthat these references are prior art in relation to the inventionsdescribed herein. In particular, the following patent applications arehereby incorporated by reference in their entireties for all purposes:PCT Application No. PCT/US2008/065563, filed Jun. 2, 2008, entitled“Production of Oil in Microorganisms”, PCT Application No.PCT/US2010/31108, filed Apr. 14, 2010, entitled “Methods of MicrobialOil Extraction and Separation”, and PCT Application No.PCT/US2009/066142, filed Nov. 30, 2009, entitled “Production of TailoredOils in Heterotrophic Microorganisms”.

What is claimed is:
 1. A microbial oil obtained from a recombinantmicroalga, wherein the fatty acid profile of the oil comprises at least40% saturated fatty acid having C10, C12 and C14, and the oil furthercomprises sterols of the recombinant microalga, wherein the recombinantmicroalga has been genetically engineered to express at least one lipidpathway enzyme at an altered level compared to a non-engineeredmicroalga, the lipid pathway enzyme being selected from the groupconsisting of a desaturase, a ketoacyl synthase, and an acyl-ACPthioesterase.
 2. The microbial oil of claim 1, wherein the at least onelipid pathway enzyme is an endogenous lipid pathway enzyme that isexpressed at a lower level compared to the non-engineered microalga. 3.The microbial oil of claim 2, wherein the at least one endogenous lipidpathway enzyme is a desaturase.
 4. The microbial oil of claim 3, whereinthe at least one endogenous lipid pathway enzyme is fatty acyldesaturase enzyme.
 5. The microbial oil of claim 3, wherein the at leastone endogenous lipid pathway enzyme is a stearoyl ACP desaturase-enzyme.6. The microbial oil of claim 2, wherein the at least oneendogenouslipid pathway enzyme is an acyl-ACP thioesterase.
 7. The microbial oilof claim 2, wherein the at least one endogenous lipid pathway enzyme isa ketoacyl synthase.
 8. The microbial oil of claim 7, wherein the atleast one endogenous lipid pathway enzyme is a ketoacyl synthase IIenzyme.
 9. The microbial oil of claim 7, wherein the at least oneendogenous lipid pathway enzyme is a ketoacyl synthase I enzyme.
 10. Themicrobial oil of claim 7, wherein the at least one endogenous lipidpathway enzyme is a ketoacyl synthase IV enzyme.
 11. The microbial oilof claim 1, wherein the at least one lipid pathway enzyme is expressedat a higher level compared to the non-engineered microalga.
 12. Themicrobial oil of claim 11, wherein the at least one lipid pathway enzymeis a ketoacyl synthase.
 13. The microbial oil of claim 1, wherein thefatty acid profile of the oil has not been altered by further processingthe microbial oil by one or more of esterification, distillation,fractionation, crystallization, and precipitation.
 14. The microbial oilof claim 1, wherein the fatty acid profile of the oil comprises at least70% saturated fatty acid.
 15. The microbial oil of claim 1, wherein thefatty acid profile of the oil comprises at least 4% C10.
 16. Themicrobial oil of claim 1, wherein the fatty acid profile of the oilcomprises at least 10% C12.
 17. The microbial oil of claim 1, whereinthe fatty acid profile of the oil comprises at least 10% C14.
 18. Themicrobial oil of claim 1, wherein the fatty acid profile of the oilcomprises at least 40% C14.
 19. The microbial oil of claim 1, whereinthe fatty acid profile of the comprises at least 50% total combinedamounts of C10:0, C12:0 and C14:0.
 20. The microbial oil of claim 1,wherein the microbial oil is Prototheca oil or Chlorella oil.
 21. Themicrobial oil of claim 20, wherein the microbial oil is Protothecamoriformis oil or Chlorella protothecoides oil.
 22. A blendedcomposition comprising a microbial oil of claim 1 and an oil from soy,rapeseed, canola, palm, palm kernel, coconut, corn, waste vegetable,Chinese tallow, olive, sunflower, cottonseed, chicken fat, beef tallow,porcine tallow, microalgae, macroalgae, microbes, Cuphea, flax, peanut,choice white grease, lard, Camelina sativa, mustard seed, cashew nut,oats, lupine, kenaf, calendula, help, coffee, linseed (flax), hazelnut,euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice,tung tree, cocoa, copra, opium poppy, castor beans, pecan, jojoba,macadamia, Brazil nuts, avocado, petroleum, or a distillate fraction ofany of the preceding oils.
 23. The oil of claim 1, wherein the oil has aC12:C14 fatty acid ratio of at least 5:1.
 24. The oil of claim 1,wherein the oil has a C12-C14 level of over 49%.
 25. The microbial oilof claim 1, wherein the fatty acid profile of the oil comprises at least70% total combined amounts of C10:0, C12:0 and C14:0.